Use of consensus sequence as vaccine antigen to enhance recognition of virulent viral variants

ABSTRACT

The invention provides consensus sequences for hepatitis C virus 1a and 1b. Also provided are non-synonymous changes for each residue of the consensus sequences. These sequences are useful as compositions or vaccines for prophylactic use or treating HCV-infected individuals. Also provided are methods for lessening the chances for a HCV-infected individual to enter a chronic phase of infection and methods of diagnosing an individual with HCV 1a or HCV 1b infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit and priority of U.S. Provisional Application Ser. No. 60/648,550, filed Jan. 31, 2005, and Provisional Application Ser. No. 60/648,877, filed Feb. 2, 2005, both of which are herein incorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under DA-11880, DK-57998 and AI-40035 awarded by the PHS. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to consensus sequences for hepatitis C virus and uses thereof. The invention also relates to immunogenic epitopes of hepatitis C virus and uses thereof. The invention also relates to methods of prophylaxis, treating, and diagnosing individuals infected with or exposed to hepatitis C virus.

BACKGROUND OF THE INVENTION

Currently, it is estimated there are about 270 to 300 million people worldwide who are infected with hepatitis C virus (HCV), 2.7 to 4 million of those are in the United States. In industrialized countries, HCV accounts for 20% of cases of acute hepatitis, 70% of cases of chronic hepatitis, 40% of cases of end-stage cirrhosis, 60% of cases of hepatocellular carcinoma and 30-40% of liver transplants. The incidence of new symptomatic infections of HCV has been estimated to be 13 cases/100,000 persons annually. For every one person that is infected with the AIDS virus, there are more than four infected with HCV. Currently, 8,000 to 10,000 deaths each year are a result of Hepatitis C (updated Jan. 31, 2006). The CDC (Center For Disease Control) estimates that there are up to 30,000 new HCV infections in the U.S. every year.

About 80% of HCV-infected individuals have no signs or symptoms. In others, the symptom include: jaundice, fatigue, dark urine, abdominal pain, loss of appetite, and nausea. For 55-85% of infected individuals, over the long term, HCV infection persists and become a chronic infection. Of the chronically infected individuals, about 30% develop liver disease.

Hepatitis C virus has six different genotypes. The most prevalent types circulating in the Western countries are sub-genotypes 1a and 1b. In a person with chronic infection, hepatitis C virus reproduces up to 10¹² virion each day. Neumann, et al. Science 282:103-107 (1998). This rate of reproduction exceeds the rate of reproduction for human immunodeficiency virus (HIV) by an order of magnitude. The reproduction rate of HCV coupled with the lack of proofreading function by the HCV RNA polymerase results many mutations in HCV sequences.

Treatments for hepatitis C include interferon and ribavirin, both of which are licensed for the treatment of persons with chronic hepatitis C. Interferon can be taken alone or in combination with ribavirin. Combination therapy, using pegylated interferon and ribavirin, is currently the treatment of choice. Combination therapy can get rid of the virus in up to 5 out of 10 persons for genotype 1 and in up to 8 out of 10 persons for genotype 2 and 3.

There are no known vaccines for the prevention of hepatitis C virus infection. Until the present inventors made their discovery herein, the reason as to why individuals with chronic stage HCV infection had weak and ineffective immune responses was poorly understood. The inventors and others have demonstrated that most, if not all of the HCV sequences obtained from persons with HCV infection contain epitope escape variants. As such, the inventors have made a novel and useful discovery that describes, inter alia, the reasons for the observed genetic changes taking place during infection of a human host. These include the tendency of the virus to mutate towards a consensus sequence that is optimal for viral replication and fitness, and the competing tendency of the virus to mutate away from sequences that induce effective immunological responses in particular human hosts. This discovery has important implications for vaccine design.

Several computational alternatives to isolate-based vaccine design exist. One approach is reconstruction of the most recent common ancestor (MRCA) sequence. In this type of analysis, the ancestral state is an estimate of the actual sequence that existed in the past (i.e., it comes directly from the reconstructed history). See Science, 299:1515-1518 (2003). Another type of computational analysis is a center of the tree (COT) approach. The COT approach identifies a point on the unrooted phylogeny, where the average evolutionary distance from that point to each tip on the phylogeny is minimized. Advocates of this approach state that because the COT is a point on the phylogeny, the estimated COT sequence will have the same advantages as the estimated ancestral sequence. See, for example, U.S. Application 2005/0137387 A1. However, this COT approach is sufficiently complex that reducing it to practice for a large and heterologous data set is not practical with technology; specifically, the phylogenetic methods cannot address a sparse data set like the one for HCV, wherein most of the data for any individual sequence are missing. In addition, the premise of the COT approach is that when the phylogenetic tree is unbalanced (dominated by a particular lineage), the COT approach proposed therein provides a more representative sequence than the ancestral sequence. However, the HCV tree has been shown, by the inventors and others, to be balanced and star-like (see Ray S C et al, J Exp Med 2005 Jun. 6; 201(11):1753-9 and Salemi M and Vandamme A, J Mol Evol 2002; 54:62-70). Overall, the MRCA and COT approaches are impractical for application to the HCV sequence database, and their primary justification does not apply.

A third type of computational analysis is the consensus sequence approach. Because the consensus sequence is composed of the amino acid most commonly observed at each position, it likely represents the most fit state of the virus. Thus, effective evasion of the immune response by selection of a sequence divergent from consensus may result in a less fit virus from a replicative standpoint. The consensus sequence approach favors heavily sampled sublineages and deemphasizes outliers. The consideration of an unbalanced phylogenetic tree is not important for HCV, because the phylogeny is balanced (star-like). As such, the approaches disclosed herein are far more straightforward than the other types of computational analysis. Furthermore, these approaches can use the entire data set for HCV. One advantage of the consensus sequence is that it minimizes the genetic differences between vaccine strains and contemporary isolates, effectively reducing the extent of diversity by half, and thus it may have enhanced potential for eliciting cross-reactive responses.

A computational method is therefore needed to generate a sequence for use in vaccines that more broadly represents circulating strains, and also restores the immunogenic forms of HCV epitopes. Currently, there is a need for a method to effectively treat individuals who are infected with HCV or exposed to HCV. With the decline in an infected individual's immune system as he/she enters chronic phase, it would be highly desirable to lessen that individual's chances of progressing to the chronic phase by administering a form of treatment during the acute phase of infection. Most chronically infected people are ineligible for the currently available therapies. A vaccine could be used to enhance responses to currently available or future therapies. Further, there is a need for a prophylaxis of HCV infection. The invention disclosed herein meets all these needs and provides even more beneficial uses.

All references, patent, and patent applications cited in this patent application are herein incorporated by reference, each in its respective entirety for all purposes.

BRIEF SUMMARY OF THE INVENTION

The invention provides for an isolated nucleic acid encoding an HCV polyprotein or a fragment thereof wherein the HCV polyprotein comprises the consensus sequence 1a (SEQ ID NO: 1). In one embodiment, the invention is a nucleic acid wherein the encoded HCV polyprotein or fragment thereof comprises one or more of the non-synonymous changes shown in Table 5.

In one aspect, the invention is an isolated nucleic acid encoding an HCV polyprotein or a fragment thereof wherein the HCV polyprotein comprises the consensus sequence 1b (SEQ ID NO: 2). In one embodiment, the invention is a nucleic acid wherein the encoded HCV polyprotein or fragment thereof comprises one or more of the non-synonymous changes shown in Table 6.

In another aspect, the invention is an isolated HCV protein having the amino acid sequence comprising the consensus sequence 1a (SEQ ID NO:1) or a fragment thereof. In another aspect, the invention is an isolated HCV protein having the amino acid sequence comprising the consensus sequence 1b (SEQ ID NO:2) or a fragment thereof. In another aspect, the invention is an isolated HCV 1a core protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 5. In another aspect, the invention is an isolated HCV 1a E1 protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 5. In another aspect, the invention is an isolated HCV 1a E2 protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 5. In another aspect, the invention is an isolated HCV 1a p7 protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 5. In another aspect, the invention is an isolated HCV 1a NS2 protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 5. In another aspect, the invention is an isolated HCV 1a NS3 protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 5. In another aspect, the invention is an isolated HCV 1a NS4a protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 5. In another aspect, the invention is an isolated HCV 1a NS4b protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 5. In another aspect, the invention is an isolated HCV 1a NS5a protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 5. In another aspect, the invention is an isolated HCV 1a NS5b protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 5.

In yet another aspect, the invention is an isolated HCV 1b core protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 6. In another aspect, the invention is an HCV 1b E1 protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 6. In another aspect, the invention is an HCV 1b E2 protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 6. In another aspect, the invention is an HCV 1b p7 protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 6. In another aspect, the invention is an HCV 1b NS2 protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 6. In another aspect, the invention is an HCV 1b NS3 protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 6. In another aspect, the invention is an HCV 1b NS4a protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 6. In another aspect, the invention is an HCV 1b NS4b protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 6. In another aspect, the invention is an HCV 1b NS5a protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 6. In another aspect, the invention is an HCV 1b NS5b protein sequence or fragments thereof comprising the consensus sequence or non-synonymous changes as shown in Table 6. In another aspect, the invention is an consensus protein which comprises at least 5 contiguous amino acids of a hepatitis C 1a virus.

The invention also provides for an isolated expression construct comprising the following operably linked elements: transcription promoter and a nucleic acid encoding an HCV consensus protein or a fragment thereof. In another aspect, the invention is an isolated consensus protein or fragment thereof from Hepatitis C 1a virus which is immunogenic. In another aspect, the invention is an isolated consensus protein which comprises at least 5 contiguous amino acids of a hepatitis C 1b virus. In another aspect, the invention is an isolated expression construct comprising the following operably linked elements: transcription promoter and a nucleic acid encoding the consensus protein of claim 30 or a fragment thereof. In another aspect, the invention is an isolated consensus protein or fragment thereof from Hepatitis C 1b virus which is immunogenic. In another aspect, the invention is an isolated nucleic acid encoding an HCV epitope capable of eliciting an immunogenic response in an individual wherein the sequence of the epitope is selected from any of the epitopes listed in Table 7. In another aspect, the invention is an isolated HCV epitope capable of eliciting an immunogenic response in an individual wherein the sequence of the epitope is selected from any of the epitopes listed in Table 7.

The invention also provides for composition comprising at least one HCV protein or a fragment thereof encoded by a polynucleotide. In another aspect, the invention is a composition comprising at least one HCV protein or a fragment thereof encoded by a polynucleotide. In another aspect, the invention is a composition comprising at least one HCV protein or a fragment thereof. In another aspect, the invention is a composition comprising at least one nucleic acid sequence for HCV consensus sequence. In another aspect, the invention is a composition comprising at least one nucleic acid sequence for HCV consensus sequence. In another aspect, the invention is a composition comprising at least one nucleic acid sequence which codes for an HCV protein or a fragment thereof.

The invention also provides for a vaccine comprising all or a portion of consensus sequence 1a (SEQ ID NO:1). In one embodiment, the vaccine comprises a non-synonymous change at a modal consensus sequence. In another aspect, the invention is a vaccine comprising all or a portion of consensus sequence 1b (SEQ ID NO:2). In one embodiment, the vaccine comprises wherein there is a non-synonymous change at a modal consensus sequence.

The invention also provides for a method of identifying an immunogen for use as a vaccine comprising: a) Obtaining a sequence of HCV that is derived from a subject and is longer than 500 nucleotides; b) Obtaining the primary open reading frame of the sequence; c) Removing sequences that contain more than 1% ambiguous sites or more than 1 frameshift; d) Converting terminal “gap” characters to “missing”; e) Removing sequences that are redundant by identifying identical sequences and checking related publications and removing linked sequences; f) Generating predicted polyprotein sequences by using standard eukaryotic genetic code; and g) Identifying majority-rule consensus sequence for each subtype to identify modal amino acid residue at each site.

The invention also provides for a method of inducing or augmenting an immune response against hepatitis C virus comprising administering an effective amount of any one of the vaccines recited disclosed herein. The invention also provides for a method for protecting an individual from hepatitis C virus infection comprising administering an effective amount of any one of the vaccines disclosed herein. The invention also provides for a method of lessening the probability that a HCV-infected individual will enter a chronic phase of hepatitis C infection comprising administering an effective amount of any one of the vaccine disclosed herein.

The invention also provides for a method for diagnosing an individual infected with hepatitis C virus 1a comprising: a. obtaining a biological sample from the individual and b. using PCR primers to consensus sequences of HCV 1a to amplify nucleic acids in the biological sample to determine if the individual has been infected with hepatitis C virus 1a. The invention also provides for a method for diagnosing an individual infected with hepatitis C virus 1b comprising: a. obtaining a biological sample from the individual and b. using PCR primers to consensus sequences of HCV 1b to amplify nucleic acids in the biological sample to determine if the individual has been infected with hepatitis C virus 1b. The invention also provides for a kit comprising a HCV vaccine and instructions for the administration thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows fluctuating HCV RNA level during acute infection. Spontaneous clearance (SC) occurred in one subject, and persistence developed in seven others. A solid triangle (▴) indicates detectable HCV RNA. An open triangle (Δ) indicates an HCV RNA level of less than 50 IU/ml. The number in the bottom right corner of each panel is the subject number. A gray inverted triangle (▾) indicates that IFN-γ ELISPOT analysis of T cell responses was performed at that time point. Subject 28 entered the study antibody negative and HCV RNA positive so that the time of infection is estimated using the average time from infection to seroconversion.

FIG. 2 shows amino acid substitutions and epitopes recognized during the first 6 months of HCV infection for five subjects. The map at the top of the figure indicates the region of the HCV polyprotein sequenced. For each study subject, horizontal lines represent the sequences obtained at initial viremia (t.sub.0), six months after viremia was first detected (t₆), and 12 months after viremia was first detected (t₁₂). Thin vertical lines represent amino acid substitutions. Shorter, thicker vertical lines indicate recognized epitopes, below which the t₀ and t₆ sequences of the epitope are shown. The sequences shown for subject 18 are from left to right: SEQ ID NOs: 7, 8, and 3. The sequences shown for subject 17 are from left to right SEQ ID NOs: 9, 10, 11, 12, and 3. The sequences shown for subject 28 are from left to right: SEQ ID NOs: 13, 14, 15, 16, 17, 18, 4, and (5 and 6). The sequences shown for subject 29 are from left to right: SEQ ID NOs: 20, 17, and 21.

FIG. 3 shows amino acid substitutions in epitopes and the effect on T cell responses. Peptide sequences observed to vary between to (circle, initial viremia) and t₆ (triangle, 6 months after onset of viremia) were used as antigens in IFN-.gamma. ELISPOT, using PBMC (filled symbols) or T cell lines generated from PBMC (open symbols) obtained at t₆ as effectors. (A) Loss of recognition: For 4 of 10 peptide pairs, recognition of the t₆ variant peptide was dramatically reduced. Sequences shown are from left to right: SEQ ID NOs: 15, 14, 16, and 9. (B) Decreased recognition: For 4 of 10 peptide pairs, recognition of the t₆ variant peptide was reduced more than 2-fold but less than 20-fold at least two concentrations of peptide. Sequences shown are from left to right: SEQ ID NOs: 10, 11, 4, and 22 (C) Comparable recognition: For 2 of 10 pairs of peptides, recognition of the t₆ variant was not reduced more than 2-fold relative to the to peptide at more than one concentration tested. Sequences shown are from left to right: SEQ ID NOs: 19 and 18.

FIG. 4 shows similar recognition patterns using lines and PBMC. IFN-γ ELISPOT responses for both a T cell line (open symbols) and for PBMC (closed symbols) from which the line was generated using the peptide KLVALGINAV are shown using the same antigens. The peptide representing the to sequence is KLVALGINAV (circles; SEQ ID NO: 3) and the peptide representing the t₆ sequence is KLVAMGINAV (triangles; SEQ ID NO: 4)). As shown for this peptide pair, responses for PBMC and T cell lines using five distinct peptide t.sub.0/t₆ pairs were consistently similar aside from the expected differences in the proportion of responding cells (note difference in axes).

FIG. 5 shows results that show recognition patterns persist with time. To rule out the subsequent development of T cell responses to the t₆ HCV sequence, IFN-γ ELISPOT testing for recognition of the t₆ peptides demonstrating escape was also performed for subjects 17 and 28 using PBMC obtained approximately 12, 18, 24, and, for subject 17, 36 months following initial infection. The number in the bottom right corner of each panel is the subject number. Patterns of recognition persisted over time and recognition of the t₆ peptides (▴) declined in parallel with the decline in recognition of the to peptides (●) that occurred with prolonged infection. From top row to bottom row of panels, going from left to right in each row, the sequences shown are SEQ ID NOs: 9, 15, 10, 16, 11, 18, 14, and 19.

FIG. 6 shows a phylogenetic analysis of HCV 18-22 years after common-source outbreak. (A) Phylogenetic tree of 5.2 kb sequence alignment placing outbreak clade (*) in context with reference sequences for all major subtypes. (B) Detailed analysis of the outbreak clade, using 10 cDNA clones from each study subject to obtain the sequence of a 698 nt region spanning the E1/E2 junction. The label “inoculum” indicates twenty clones from inoculum source plasma (10 each from 2 specimens), and a full-length clone (Genbank Accession No. AF313916) obtained in an independent study of this material using smaller amplicons. For both trees, numbers at nodes are bootstrap values, indicating the percentage of 1000 permuted trees that supported the presence of that node. Bootstrap support was 100% for each of the major clades in panel A; in panel B, only bootstrap values greater than 80% are shown, and values for nodes within a study subject's clade were omitted for clarity. Boxes highlight 2 subjects whose sequences were segregated into two separate clades.

FIG. 7 shows HCV divergence and convergence following a common-source outbreak. The upper panel shows a sliding-window analysis of nonsynonymous (lighter curve, with lower mean variability) and synonymous (darker curve, with higher mean variability) variation, calculated by comparing the mean pairwise distance between the inoculum (2 cDNA clones obtained from two inoculum source plasma specimens obtained one week apart in 1977) to 44 clones (2 per study subject) obtained from chronically-infected women 18-22 years after exposure, in a sliding window 20 codons wide, moving in 1-codon increments, generated using VarPlot. Horizontal bars indicate average distance for each gene region. The lower panel shows detail of variability in HVR1 (the region indicated by dashed lines in the upper panel), indicating position relative to the H77 polyprotein (residues 384 to 410 in SEQ ID NO: 1 and 2). The first three rows show the subtype 1b reference sequences, inoculum sequences (20 clones from 2 plasma specimens obtained one week apart), and 220 recipient sequences (10 per study subject) respectively, with the height of each single-letter amino acid code proportional to its frequency. The fourth row shows differences between the amino acid frequencies in the recipient sequences versus the inoculum sequences as a type 2 logo, in which the height of each amino acid is determined by the log₂ relative risk of observing it, with the scale indicated. Empty spaces indicate a distribution highly similar to the inoculum distribution, because the logarithm of a relative risk of one is zero.

FIG. 8 shows escape versus reversion in the presence versus absence of the restricting HLA allele. (A) Amino acid alignment of a region in NS3 (positions 1388 to 1431 relative to strain H, GenBank entry AF009606; SEQ ID NO: 1 or 2) showing sites with polymorphism. Study subjects are listed in arbitrary order. Identity to the inoculum sequence (“inoc”) is indicated by “.”. (B) Sorting the subjects by the presence of HLA A*02, G14095 substitution in the 4th position of a frequently-recognized HLA A*02-restricted epitope at 1406-1415 is limited to subjects having the HLA A*02 allele. The subtype 1b consensus sequence for this epitope is shown above the alignment, and has been shown to be recognized as readily as the prototype (subtype 1a) KLVALGINAV sequence (23; SEQ ID NO: 3). Subject AD17 (HLA A*01, A*11) had G1409D substitution, the impact of which on recognition is unknown. (C) Variation resulting in reversion to a HLA B*08-restricted epitope. Sorting the subjects by presence of HLA B*08, R1397K substitution in the 3rd position of a frequently-recognized HLA B*08-restricted epitope at 1395-1403 is limited to subjects lacking the HLA B*08 allele. The inoculum sequence differs from the prototypical epitope (HSKKKCDEL; SEQ ID NO: 18) at the 3rd position. Reversion to consensus (and the prototype epitope) occurred only in study subjects lacking the HLA B*08 allele.

FIG. 9 shows HCV consensus sequence 1a (SEQ ID NO:1).

FIG. 10 shows HCV consensus sequence 1b (SEQ ID NO:2).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides consensus sequences for HCV 1a sub-genotype (or subtype) and HCV 1b subtype. The invention further provides details on which residues of the consensus sequence have known amino acids substitutions. In addition, the invention further provides epitopes useful for inducing immune responses to HCV. The consensus sequence, its variants, and epitopes provides for vaccines for prophylaxis against and treatment of chronic HCV infection. The vaccines further provide a method for inducing an immune response against HCV. Further, the invention provides methods for preventing an individual's entrance into a chronic phase of HCV. The invention also provides methods of diagnosis of HCV 1a and 1b. The invention further provides for kits for use in prophylaxis and treatment of HCV.

DEFINITIONS

All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

An “B cell epitope” is a term well-understood in the art and means any chemical moiety which exhibits specific binding to an antibody. An “epitope” can also comprise an antigen, which is a moiety or molecule that contains an epitope, and, as such, also specifically binds to antibody.

A “T cell epitope” means a component or portion thereof for which a T cell has an antigen-specific specific binding site, the result of binding to which activates the T cell.

The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases.

An “individual” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats.

A “biological sample” encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term “biological sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.

As used herein, the singular form “a”, “an”, and “the” includes plural references unless indicated otherwise. For example, “an” epitope includes one or more epitopes.

Consensus Sequences

Hepatitis C virus mutates rapidly and has two to three times more genetic variability than HIV. Hepatitis C reproduces more than 100 billion times a day, about 100 times faster than HIV. The inventors have discovered that instead of random changes, as conventional wisdom surmised, the HCV changes in a “Darwinian” manner. That is, the viral genome changes in a way to make the virus more reproductively fit in the face of each individual immune system it encounters. The inventors have discovered that the HCV genome changed in a manner to evade the individual's immune system in regions subject to effective T cell immune responses; and in regions where there was no effective T cell response due to lack of HLA epitopes, the HCV genome naturally mutated toward a consensus sequence which was the viruses' most replicatively fit state.

The HCV polyprotein is expressed as a single open reading frame, and is then processed by host (structural genes through the p7/NS2 junction) and viral (remainder) proteases. The structure and numbering of the positions in the HCV genome and polyprotein are based by convention on the subtype 1a genome, as described at the Los Alamos National Laboratory HCV sequence database web site (URL:http://hcv.lanl.gov/content/hcv-db/MAP/landmark.html) and in a recent publication (see Simmonds P, et al. Hepatology 2005; 42:962-73). HCV 1a core protein spans residues 1 to 191. E1 protein spans residues 192 to 383. E2 protein spans residues 384 to 746. p7 protein spans residues 747 to 809. NS2 protein spans residues 810 to 1026. NS3 protein spans residues 1027 to 1657. NS4a protein spans residues 1658 to 1711. NS4b protein spans residues 1712 to 1972. NS5a spans residues 1973 to 2420. NS5b protein spans residues 2421 to 3011. For HCV 1b subtype, there is an insertion at residue 2413 of an additional residue relative to the consensus 1a sequence.

Determination of a Consensus Sequence

To determine a consensus sequence from any genotype of HCV, or other viruses, one method that can be used is as follows: a) Obtain a sequence of HCV or the virus of interest that is derived from a subject (e.g., a human) and is longer than 500 nucleotides; b) Obtain the primary open reading frame of the sequence; c) Remove sequences that contain more than 1% ambiguous sites or more than 1 frameshift; d) Convert terminal “gap” characters to “missing”; e) Remove sequences that are redundant by identifying identical sequences and checking related publications and removing linked sequences; f) Generate predicted polyprotein sequences by using standard eukaryotic genetic code; and g) Identify majority-rule consensus sequence for each subtype to identify modal amino acid residue at each site.

The methodology disclosed herein is for obtaining consensus sequences and is not the same methodology used for obtaining ancestral sequences or center of tree (COT) sequences. However, for balanced (i.e., star-like) phylogeny like that seen for HCV, all three methodologies could yield the same end result. For commentary on general consensus and ancestral states as applied to HIV, see Science Vol. 29, pp. 1515-1518 (2003). However, the methodology disclosed herein provides advantages in that it is far more straightforward to use than previously disclosed methods and can be used with the entire data set for HCV, which has never been done before.

As detailed in the Examples, by using the methodology disclosed herein consensus sequence 1a (SEQ ID NO: 1) (FIG. 9) and consensus sequence 1b (SEQ ID NO:2) (FIG. 10) were obtained. Furthermore, the invention not only discloses the consensus sequence, but also details precisely which residues have non-synonmyous (amino acid changing) changes and precisely what those changes are at a certain frequency. The consensus sequence and its amino acid substitutions (or variants) are displayed in Tables 5 and 6. The first column indicates the residue position of each consensus sequence and the remaining columns indicate the residue-frequency displayed as a pair. The frequency is displayed as a subscript of the residue in this table. Residues indicated with an “X” means that that one or more sequences in the database have errors or ambiguity at that site, resulting in a codon that does not map to the genetic code. Common reasons for this include single base deletions and multistate characters (e.g., IUPAC R for A or G; Y for C or T, etc). BioEdit translates these as “X” to indicate that an amino acid could not be assigned. Using this information, one of skill in the art can predict what substitutions, if any, can be expected at a particular residue position and if a substitution is expected, then the information provided herein allows one to know which amino acids will be expected and at what frequency. For each of the 3011 residues for HCV 1a and 3010 residues for HCV1b, there are 19 other amino acids possible for substitution. It creates an overwhelming number of possible sequences for one of skill in the art to experiment with if there were not the kind of explicit teaching provided herein. It would be impossible for one of skill in the art to predict, a priori, what the consensus sequence would be for HCV 1a and HCV 1b and which amino acids are found as possible substitutes at each of the 6021 positions possible and at what frequency these non-synonymous changes are found.

Polypeptides of HCV Consensus Sequences

The invention also provides for consensus sequences for the entire HCV polyprotein as well as for each HCV protein that has been processed by host or viral proteases. These HCV proteins include: core, E1, E2, p7, NS2, NS3, NS4a, NS4b, NS5a, and NS5b. One aspect of the invention contemplates the entire HCV polyprotein while other aspects of the invention contemplate the HCV proteins post-processing. The invention encompasses HCV proteins with non-synonymous changes as well as heterologous polypeptides.

The invention further provides a number of epitopes that have been found to be immunogenic (i.e., capable of inducing an immune response). These epitopes are disclosed in Table 7. As detailed in the Examples, some of these epitopes have been mapped systemically and found to be immunogenic. HCV has been found to mutate more frequently in the areas which comprise T cell epitopes than in areas which are not within T cell epitopes. Individuals who self-recovered from HCV infection had no changes in the sequences in the T cell epitope areas while those who progressed to chronic infection had one or more changes within the T cell epitopes. Further, in those chronically infected individuals with changes outside the T cell epitopes, many of the substitutions resulted in an amino acid that matched the consensus sequence. B cell epitopes are contained within the consensus sequences disclosed herein. Accordingly, the invention encompasses T cell epitopes as well as B cells epitopes. Accordingly, the invention encompasses polypeptides comprising these epitopes and heterologous polypeptides comprising these epitopes.

The invention also encompasses polypeptides which are fusion proteins. A fusion protein is a single polypeptide comprising regions from two or more different proteins. The regions normally exist in separate proteins and are brought together in the fusion protein. They may be linked together so that the amino acid sequence of one begins where the amino acid sequence of the other ends, or they may be linked via linker amino acids which are not normally a part of the constituent proteins. A fusion protein may contain more than one copy of any of its constituent proteins. The constituent proteins may include the entire amino acid sequences of the proteins or portions of the amino acid sequences. In one embodiment, the fusion protein of this invention are two or more HCV proteins (e.g., core, E1, E2, p7, NS2, NS3, NS4a, NS4b, NS5a, and NS5b) or fragments thereof brought together as a fusion protein. The HCV proteins can contain substitutions at various residue positions as disclosed in Tables 5 and 6. In another embodiment, a fusion protein is made from two or more epitopes disclosed herein. The epitopes can be a combination of different T cell epitopes or a combination of B cell epitopes or a combination of both T cell and B cell epitopes.

A vector can include nucleic acid coding for the fusion protein of the invention in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention can be designed for expression of the fusion proteins of the invention in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, mammalian cells in culture, or in transgenic animals. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

A way to maximize recombinant protein expression in E. coli is to express the protein in host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

The fusion protein expression vector can also be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian cells in culture, or in transgenic animals. When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and iimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

A “host cell” includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incorporation of polynucleotide molecules and/or proteins. A host cell can be any prokaryotic or eukaryotic cell. For example, fusion proteins of the invention can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.

Polynucleotides Encoding the HCV Consensus Sequences

The invention also encompasses polynucleotides encoding the HCV consensus sequences or fragments thereof. Polynucleotides coding for the epitopes disclosed herein are also encompassed by the invention. Polynucleotides coding for heterologous polypeptides and for fusion proteins are also encompassed by this invention.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney), ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir & C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991) and Short Protocols in Molecular Biology (Wiley and Sons, 1999).

The invention further provides isolated polynucleotides that encode a HCV consensus sequence or fragments thereof, as well as vectors comprising the polynucleotide and a host cell containing the vector. Such expression systems can be used in a method of producing an HCV consensus sequence polypeptide or fragments thereof, wherein the host cell is cultured and the polypeptide produced by the cultured host cell is recovered. Polynucleotides encoding consensus sequences of the invention can also be delivered to a host subject for expression of the consensus sequence by cells of the host subject.

Polynucleotides complementary to any such sequences are also encompassed by the present invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

Polynucleotides may comprise the consensus sequence (or a fragment thereof) or may comprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not diminished, relative to the original immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein. Polynucleotide variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to a polynucleotide sequence that encodes the HCV consensus sequences disclosed herein or a fragment thereof.

Two polynucleotide or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 5 contiguous positions, at least about 10 contiguous positions, at least about 15 contiguous positions, or at least about 20 contiguous positions in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

Preferably, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 5 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

Polynucleotides may be prepared using any of a variety of techniques known in the art. The nucleic acid sequence that encodes for the HCV consensus sequences disclosed herein may be obtained from publicly available databases (e.g., GenBank) or from reverse translation from the amino acid sequence of the HCV consensus sequence. Although hepatitis C virus is a RNA virus, DNA sequences (including cDNA) that code for the HCV consensus sequences are also encompassed by this invention. Such nucleic acid sequences can also be obtained as part of a genomic library or a cDNA library. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989.

Polynucleotides, polynucleotide variants, and whole viruses may generally be prepared by any method known in the art, including chemical synthesis by, for example, solid phase phosphoramidite chemical synthesis. Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (see Adelman et al., DNA 2:183, 1983). Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding an antibody, or portion thereof, provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide, as described herein. In addition, or alternatively, a portion may be administered to an individual such that the encoded polypeptide is generated in vivo (e.g., by transfecting antigen-presenting cells, such as dendritic cells, with a cDNA construct encoding the polypeptide, and administering the transfected cells to the individual).

Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends; the use of phosphorothioate or 2′O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl-, methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

Nucleotide sequences can be joined to a variety of other nucleotide sequences using established recombinant DNA techniques. For example, a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. In general, a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other elements will depend upon the desired use, and will be apparent to those of ordinary skill in the art.

Within certain embodiments, polynucleotides may be formulated so as to permit entry into a cell of a mammal, and to permit expression therein. Such formulations can be particularly useful for therapeutic purposes, for example for treating HCV infection or as a prophylaxis. In one embodiment, the invention encompasses an expression construct for expressing any portion of the consensus sequence will contain the following operably linked elements: a transcription promoter, a nucleic acid encoding all or some fragment of the consensus sequences and its possible variants disclosed herein, and a transcription terminator. Those of ordinary skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus). Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.

Additional methods of expression for vaccine purposes are disclosed below. Other formulations for therapeutic purposes include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

Uses for Consensus Sequences

The invention provides for many uses for the consensus sequences disclosed herein. In one aspect, an isolated consensus protein with the sequences disclosed herein is used as a vaccine for prophylactic and treatment purposes. In another aspect, the invention provides for the uses of HCV epitopes disclosed herein to induce or augment an immune response in an individual. In another aspect, the invention is a composition comprising at least one HCV 1a or 1b consensus protein or a fragment thereof. In yet another aspect, the invention is a pharmaceutical composition comprising at least one HCV 1a or 1b consensus protein or a fragment thereof and a suitable carrier. It is understood that this invention not only encompasses polypeptides comprising the consensus sequences but also polynucleotides coding for the polypeptides.

The consensus sequences disclosed herein can be used in its entirety or as fragments. In one embodiment, the consensus sequence comprises at least one HCV protein (e.g., core, E1, E2, p7, NS2, NS3, NS4a, NS4b, NS5a, or NS5b) or a fragment thereof. In another embodiment, the consensus protein fragment has at least 5 contiguous amino acids of either HCV 1a or HCV1b virus. In another embodiment, the consensus protein fragment has least 8 contiguous amino acids of either HCV 1a or HCV1b virus. In other embodiments, the consensus protein fragment has least 11, at least 14, at least 17, at least 20, at least 23, at least 27, at least 30, at least 33, at least 36, at least 39, at least 42, at least 45, at least 48, or at least 51 contiguous amino acids of either HCV 1a or HCV1b virus.

As the HCV mutates, the invention provided herein teaches one of skill in the art how to predict changes in the sequence. Since some of the residues have several variants, the teachings herein on the precise amino acid and the frequency with which was observed allows one of skill in the art to practice the invention without undue experimentation.

Pharmaceutical Compositions

The invention encompasses pharmaceutical compositions comprising HCV consensus sequence proteins or fragments thereof and polynucleotides encoding the same. In addition to the HCV consensus protein (or polynucleotide), the pharmaceutical composition includes a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are known in the art, and are relatively inert substances that facilitate administration of a pharmacologically effective substance. For example, an excipient can give form or consistency, or act as a diluent. Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington's Pharmaceutical Sciences 19th Ed. Mack Publishing (1995). Generally, these compositions are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these compositions are preferably combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.

Vaccines Using HCV Consensus Sequences

Several general approaches exist for making a vaccine against a virus that mutates rapidly and has multiple subtypes around the world. From an efficiency perspective, it would be ideal to be able to make one vaccine that could be effective against the same virus, even if it has mutated as it is transmitted from person to person. One general approach is to use isolates of a particular subtype to make the vaccine. However, because it is likely that each geographical area will have mutations that are clustered and make the virus phylogenetically a different clade, the isolate approach is not economically viable because country-specific vaccine efforts would be needed. Another approach, using the consensus sequence instead of the actual sequence isolated from within the infected population, has the advantages of being central and having the potential to induce or augment cross-reactive responses in infected individuals. Other considerations when making a vaccine also include whether the protein should be polyvalent or to target specific types of valencies, for example, in HIV, designing modified envelopes to enhance exposure of epitopes known to be capable of inducing broadly neutralizing antibodies. See, for example, Gaschen at el. Science 296: 2354-2360 (2002).

Use of a consensus sequence as a vaccine for HCV should take into account considerations that would stimulate responses that drive the virus to mutate to a less fit state, thereby rendering it less able to replicate, and easier to eradicate. Such considerations include co-receptor usage (if any), protein folding, and exposure of antigenic or immunogenic domains. Selection of a consensus sequence for use as a vaccine against HCV would elicit both T cell and/or B cell responses in such way to engage the immune system to eradicate the existing HCV in the infected individual.

The vaccines of the invention comprise HCV consensus sequence or a fragment thereof or a polynucleotide encoding HCV consensus sequence or a fragment thereof. For instance, the vaccine may comprise or encode the HCV polyprotein, the primary translation product, or the full-length translation product of the HCV consensus sequence. In another embodiment, the vaccine comprises the processed HCV proteins or fragments thereof. In addition to the use of consensus sequence proteins (or polynucleotides encoding those proteins), polypeptides comprising fragments of HCV consensus sequence, or polynucleotides encoding fragments of HCV consensus sequence may be used in the vaccines. The polypeptides in the vaccines or encoded by polynucleotides of the vaccines are optionally at least about 99%, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, or at least about 50% identical to the HCV consensus sequence disclosed herein.

In addition, the polynucleotides of the vaccines are optionally at least about 99%, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, or at least about 50% identical to the polynucleotides encoding the HCV consensus sequence disclosed herein.

Delivery/Expression Systems

The HCV consensus sequence vaccines can be delivered to the individual using various expression systems. A few of the possible systems are described below.

1. Mammalian Cell-Based Delivery Systems

In one embodiment of the invention, the immunogenic composition comprises a cell. The cell of the immunogenic composition comprises a consensus sequence or epitope-containing polypeptide described herein or a polynucleotide encoding an epitope-containing polypeptide described herein. Prior to delivery of the cell to a host, the cell optionally comprises an epitope from the consensus sequence on its surface (for instance, as an HLA-bound peptide). In an alternative embodiment, the cell comprises an epitope-containing polypeptide described herein and/or a polynucleotide encoding a consensus sequence or epitope-containing polypeptide described herein in the interior of the cell. In one embodiment, the immunogenic composition comprising the consensus sequence or polynucleotide encoding the consensus sequence is an eukaryotic cell, such as a mammalian cell.

In another embodiment, the immunogenic composition comprises a mammalian cell which is an antigen-presenting cell. Antigen presenting cells, as referred to herein, express at least one class I or class II MHC determinant and may include those cells which are known as professional antigen-presenting cells such as macrophages, dendritic cells and B cells. Other professional antigen-presenting cells include monocytes, marginal zone Kupffer cells, microglia, Langerhans' cells, interdigitating dendritic cells, follicular dendritic cells, and T cells. Facultative antigen-presenting cells can also be used. Examples of facultative antigen-presenting cells include astrocytes, follicular cells, endothelium and fibroblasts. In one embodiment, the immunogenic composition comprises an antigen-presenting cell that affords MHC class I antigen presentation.

The polypeptides or polynucleotides of the invention may be delivered to cells in vivo or ex vivo. The loading of antigen onto antigen-presenting cells such as dendritic cells has been achieved by different methods, including peptide pulsing (see Melero et al., Gene Therapy 7:1167 (2000). This method for administering polypeptide vaccine is accomplished by pulsing the polypeptide onto an APC or dendritic cell in vitro. The polypeptide binds to MHC molecules on the surface of the APC or dendritic cell. Prior treatment of the APCs or dendritic cells with interferon-γ can be used to increase the number of MHC molecules on the APCs or dendritic cells. The pulsed cells can then be administered as a carrier for the polypeptide.

2. Yeast-Based Delivery Systems

In another embodiment of the invention, a consensus sequence or epitope-containing polypeptide described herein, or a polynucleotide encoding the consensus sequence or epitope-containing polypeptide, is delivered to a host organism in a vaccine comprising yeast. The use of live yeast DNA vaccine vectors for antigen delivery has been reviewed recently and reported to be efficacious in a mouse model using whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens (see Stubbs et al. (2001) Nature Medicine 7: 625-29).

The use of live yeast vaccine vectors is known in the art. Furthermore, U.S. Pat. No. 5,830,463 describes particularly useful vectors and systems for use in the instant invention. The use of yeast delivery systems may be particularly effective for use in the viral vaccine methods and formulations of the invention as yeast appear to trigger cell-mediated immunity without the need for an adjuvant. Possible yeast vaccine delivery systems are nonpathogenic yeast carrying at least one recombinant expression system capable of modulating an immune response. In an alternative, heat killed yeast may also be used to express the protein antigen of interest as part of a yeast-based delivery system (e.g., GlobeImmune products).

3. Bacterial Systems

In another embodiment, the cell that comprises the consensus sequence or epitope-containing polypeptide, or polynucleotide encoding the consensus sequence or epitope-containing polypeptide, is a microbial cell. In one embodiment, the microbial cell is a bacterium, typically a mutant or recombinant bacterium. The bacterium is optionally attenuated. For instance, a number of bacterial species have been studied for their potential use as vaccines and can be used in the present invention, including, but not limited to, Shigella flexneri, E. Coli, Listeria monocytogenes, Yersinia enterocolitica, Salmonella typhimurium, Salmonella typhi or mycobacterium. In one embodiment, the bacterial vector used in the immunogenic composition is a facultative, intracellular bacteria vector. In one embodiment, the bacterium is used to deliver an epitope-containing polypeptide to antigen-presenting cells in the host organism.

The use of live bacterial vaccine vectors for antigen delivery has been reviewed (Medina and Guzman (2001) Vaccine 19: 1573-1580; and Darji et al. (2000) FEMS Immunol and Medical Microbiology 27: 341-9). Furthermore, U.S. Pat. Nos. 6,261,568 and 6,488,926 describe particularly useful systems for use in this HCV vaccine invention.

The use of live bacterial vaccine vectors can be particularly advantageous. Bacteria-mediated gene transfer finds particular advantage in genetic vaccination by intramuscular, intradermal or oral administration of plasmids which leads to antigen expression in the mammalian host-thereby offering the possibility of both antigen modification as well as immune modulation. Furthermore, the bacterial-mediated DNA vaccine provides adjuvant effects and the ability to target inductive sites of the immune system. In preferred embodiments, S. typhimurium, S. typhi, S. flexneri or L. monocytogenes are used as vehicles for trans-kingdom DNA vaccine delivery.

Furthermore, many live bacterial vaccine vectors make use of the almost unlimited coding capacity of bacterial plasmids, and broad availability of bacterial expression vectors, to express virtually any target tumor antigen of interest. The use of bacterial carriers is often associated with still other significant benefits, such as the availability of convenient direct mucosal or oral delivery. Other direct mucosal delivery systems (besides live viral or bacterial vaccine carriers) include mucosal adjuvants, viral particles, ISCOMs, liposomes, microparticles and transgenic plants. Other advantages of this technology are: low batch preparation costs, facilitated technology transfer following development of the prototype, increased shelf-life and stability in the field respect to other formulations (e.g., subunit vaccines), easy administration and low delivery costs. Taken together, these advantages make this strategy particularly suitable for vaccine programs including HCV vaccines. The carrier operationally becomes an equivalent of a subunit recombinant vaccine.

Both attenuated and commensal microorganisms have been successfully used as carriers for vaccine antigens. Attenuated mucosal pathogens which may be used in the invention include: L. monocyotgenes, Salmonella spp., V. cholorae, Shigella spp., mycobacterium, Y. enterocolitica, and B. anthracis. Commensal strains for use in the invention include: S. gordonii, Lactobacillus spp., and Staphylococcus ssp. The background of the carrier strain used in the formulation, the type of mutation selected to achieve attenuation, and the intrinsic properties of the immunogen can be used in optimizing the extent and quality of the immune response elicited. The general factors to be considered to optimize the immune response stimulated by the bacterial carrier include carrier-related factors including: selection of the carrier; the specific background strain, the attenuating mutation and the level of attenuation; the stabilization of the attenuated phenotype and the establishment of the optimal dosage. Other considerations include antigen-related factors such as: instrinsic properties of the antigen; the expression system, antigen-display form and stabilization of the recombinant phenotype; co-expression of modulating molecules and vaccination schedules.

Descriptions of exemplary bacterial vaccine vectors follows, including references which demonstrate the availability in the art of the knowledge required to generate recombinant bacterial vaccine vectors that express the antigen of choice and can be used in immunogenic compositions. Each of the following references in incorporated herein in its entirety.

For instance, Salmonella typhimurium could be used as a bacterial vector in the immunogenic compositions of the invention. Use of this type of bacteria as an effective vector for a vaccine has been demonstrated in the art. For instance, the use of S. typhimurium as an attenuated vector for oral somatic transgene vaccination has been described (see Darji et al. (1997) Cell 91: 765-775; and Darji et al. (2000) FEMS Immun and Medical Microbiology 27:341-9). Indeed most knowledge on bacteria-mediated gene transfer has been acquired using attenuated S. typhimurium as carrier. Two metabolically attenuated strains that have been used include S. typhimurium aroA, which is unable to synthesize aromatic amino acids, and S. typhimurium 22-11, which is defective in purine metabolism. Several antigens have been expressed using these carriers: originally, listeriolysin and actA (two virulence factors of L. monocytogenes) and beta-galactosidase (beta-gal) of E. coli were successfully tested. Cytotoxic and helper T cells as well as specific antibodies could be detected against these antigens following oral application of a single dose of the recombinant salmonella. In addition, immunization with salmonella carrying a listeriolysin-encoding expression plasmid elicited a protective response against a lethal challenge with L. monocytogenes. Interestingly, this protection was observed in the lung although the vaccine was administered orally. Oral transgene vaccination methodology has now been extended to include protective responses in herpes simplex virus 2 and hepatitis B infection models, with cell-mediated immune responses detected at the mucosal level.

In another embodiment, the immunogenic compositions of the present invention optionally comprise Shigella flexneri as a delivery vehicle. S. flexneri represents the prototype of a bacterial DNA transfer vehicle as it escapes from the vacuole into the cytosol of the host cell. Several attenuated mutants have been used successfully to transfer DNA to cell lines in vitro. Auxotrophic strains were defective in cell-wall synthesis (see Sizemore et al. (1995) Science 270:299-302; and dapB (see Courvalin et al. (1995) C R Acad Sci Ser III, 318:1207-12), synthesis of aromatic amino acids (aroA (see Powell et al. (1996) Vaccines 96: Molecular Approaches to the Control of Infectious Disease; Cold Spring Harbor Laboratory Press) or synthesis of guanine nucleotides (guaBA (see Anderson et al. (2000) Vaccine 18: 2193-2202).

In still another embodiment, the immunogenic compositions of the present invention comprise Listeria monocytogenes (Portnoy et al, Journal of Cell Biology, 158:409-414 (2002); Glomski et al., Journal of Cell Biology, 156:1029-1038 (2002)). Strains of Listeria monocytogenes have recently been developed as effective intracellular delivery vehicles of heterologous proteins providing delivery of antigens to the immune system to induce an immune response to clinical conditions that do not permit injection of the disease-causing agent, such as cancer (U.S. Pat. No. 6,051,237 Paterson; Gunn et al., J. Immun. 167:6471-6479 (2001); Liau, et al., Cancer Research, 62: 2287-2293 (2002)) and HIV (U.S. Pat. No. 5,830,702 Portnoy & Paterson). A recombinant L. monocytogenes vaccine expressing a lymphocytic choriomeningitis virus (LCMV) antigen has also been shown to induce protective cell-mediated immunity to the antigen (Shen et al., PNAS, 92: 3987-3991 (1995). The ability of L. monocytogenes to serve as a vaccine vector has been reviewed (Wesikirch, et al., Immunol. Rev. 158:159-169 (1997)).

As a facultative intracellular bacterium, L. monocytogenes elicits both humoral and cell-mediated immune responses. Following entry of the Listeria into a cell of the host organism, the Listeria produces Listeria-specific proteins that enable it to escape from the phagolysosome of the engulfing host cell into the cytosol of that cell. Here, L. monocytogenes proliferates, expressing proteins necessary for survival, but also expressing heterologous genes operably linked to Listeria promoters. Presentation of peptides of these heterologous proteins on the surface of the engulfing cell by MHC proteins permits the development of a T cell response. Two integration vectors which are particularly useful for introducing heterologous genes into the bacteria for use as vaccines include pPL1 and pPL2 as described in Lauer et al., Journal of Bacteriology, 184: 4177-4186 (2002).

Attenuated forms of L. monocytogenes have been produced. The ActA protein of L. monocytogenes is sufficient to promote the actin recruitment and polymerization events responsible for intracellular movement. A human safety study has reported that administration of an actA/plcB-deleted attenuated form of Listeria monocytogenes caused no serious sequelae in adults (Angelakopoulos et al., Infection and Immunity, 70:3592-3601 (2002)).

Another possible delivery system is based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. In these microbes, for example L. monocytogenes, genes required for nucleotide excision repair (uvrAB) are removed, rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants can be blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population is still able to express its genes, synthesize and secrete proteins. See Brockstedt et al Nat. Med. 2005 August; 11(8):853-60 and US 2004/0197343 A1. Other systems that can be used include those taught in US 2004/0228877 A1, US 2005/0281783 A1, and US 2005/0249748 A1.

4. Viral-Based Delivery Systems

In another embodiment of the invention, the immunogenic composition comprising the consensus sequence or epitope-containing polypeptide and/or the polynucleotide encoding the consensus sequence or epitope-containing further comprises a viral vector. The viral vector will typically comprise a highly attenuated, non-replicative virus. Vaccinia variants, avipoxviruses, adenoviruses, polio viruses, influenza viruses, and herpes viruses can all be used as delivery vectors in conjunction with the present invention.

Formulations

Compositions comprising any of the consensus sequences described herein are also provided. In some embodiments, the compositions are pharmaceutical compositions. In some embodiments, the compositions are immunogenic compositions. The pharmaceutical compositions optionally comprise a pharmaceutically acceptable carrier or adjuvant. In some embodiments, the compositions are vaccine compositions (i.e., vaccines). The vaccine compositions optionally comprise a pharmaceutically acceptable carrier or adjuvant.

The vaccine compositions of the present invention can be used to stimulate an immune response in an individual. The formulations can be administered to an individual by a variety of administration routes. Methods of administration of such a vaccine composition are known in the art, and include oral, nasal, intravenous, intradermal, intraperitoneal, intramuscular, intralymphatic, percutaneous, scarification, and subcutaneous routes of administration, as well as intradermally by gene gun wherein gold particles coated with DNA may be used in the gene gun and any other route that is relevant for an infectious disease.

The vaccine compositions may further comprise additional components known in the art to improve the immune response to a vaccine, such as adjuvants, T cell co-stimulatory molecules, or antibodies, such as anti-CTLA4. The invention also includes medicaments comprising the pharmaceutical compositions of the invention. An individual to be treated with such vaccines, is any vertebrate, preferably a mammal, including domestic animals, sport animals, and primates, including humans. The vaccine can be administered as a prophylactic or for treatment purposes.

Vaccine formulations are known in the art. Known vaccine formulations can include one or more possible additives, such as carriers, preservatives, stabilizers, adjuvants, antibiotics, and other substances. Preservatives, such as thimerosal or 2-phenoxy ethanol, can be added to slow or stop the growth of bacteria or fungi resulting from inadvertent contamination, especially as might occur with vaccine vials intended for multiple uses or doses. Stabilizers, such as lactose or monosodium glutamate (MSG), can be added to stabilize the vaccine formulation against a variety of conditions, such as temperature variations or a freeze-drying process. Adjuvants, such as aluminum hydroxide or aluminum phosphate, are optionally added to increase the ability of the vaccine to trigger, enhance, or prolong an immune response. Additional materials, such as cytokines, chemokines, and bacterial nucleic acid sequences, like CpG, are also potential vaccine adjuvants. Antibiotics, such as neomycin and streptomycin, are optionally added to prevent the potentially harmful growth of germs. Vaccines may also include a suspending fluid such as sterile water or saline. Vaccines may also contain small amounts of residual materials from the manufacturing process, such as cell or bacterial proteins, egg proteins (from vaccines that are produced in eggs), DNA or RNA, or formaldehyde from a toxoiding process. Formulations may be re-suspended or diluted in a suitable diluent such as sterile water, saline, isotonic buffered saline (e.g. phosphate buffered to physiological pH), or other suitable diluent.

The consensus sequence vaccine is optionally administered to a host in a physiologically acceptable carrier. Optionally, the vaccine formulation further comprises an adjuvant. Useful carriers known to those of ordinary skill in the art include, e.g., citrate-bicarbonate buffer, buffered water, 0.4% saline, and the like. In some embodiments, the vaccine compositions are prepared as liquid suspensions. In other embodiments, the vaccine compositions comprising the consensus sequence strains are lyophilized (i.e., freeze-dried). The lyophilized preparation can then be combined with a sterile solution (e.g., citrate-bicarbonate buffer, buffered water, 0.4% saline, or the like) prior to administration.

Viral vectors can be used to administer polynucleotides encoding a polypeptide comprising one or more HCV consensus sequences or polynucleotides encoding a HCV epitope or any fragment thereof. Such viral vectors include vaccinia virus and avian viruses, such as Newcastle disease virus. Others may be used as are known in the art.

Naked DNA can be injected directly into the host to produce an immune response. Such naked DNA vaccines may be injected intramuscularly into human muscle tissue, or through transdermal or intradermal delivery of the vaccine DNA, typically using biolistic-mediate gene transfer (i.e., gene gun). Recent reviews describing the gene gun and muscle injection delivery strategies for DNA immunization include Tuting, Curr. Opin. Mol. Ther. (1999) 1: 216-25, Robinson, Int. J. Mol. Med. (1999) 4: 549-55, and Mumper and Ledbur, Mol. Biotechnol. (2001) 19: 79-95. Other possible methods for delivering plasmid DNA include electroporation and iontophoreses.

Another possible gene delivery system comprises ionic complexes formed between DNA and polycationic liposomes (see, e.g., Caplen et al. (1995) Nature Med. 1: 39). Held together by electrostatic interaction, these complexes may dissociate because of the charge screening effect of the polyelectrolytes in the biological fluid. A strongly basic lipid composition can stabilize the complex, but such lipids may be cytotoxic. Other possible methods for delivering DNA include electroporation and iontophoreses.

The use of intracellular and intercellular targeting strategies in DNA vaccines may further enhance the effect of HCV vaccine compositions. Previously, intracellular targeting strategies and intercellular spreading strategies have been used to enhance MHC class I or MHC class II presentation of antigen, resulting in potent CD8+ or CD4+ T cell-mediated antitumor immunity, respectively. For example, MHC class I presentation of a model antigen, HPV-16 E7, was enhanced using linkage of Mycobacterium tuberculosis heat shock protein 70 (HSP70) (Chen, et al., (2000), Cancer Research, 60: 1035-1042), calreticulin (Cheng, et al., (2001) J Clin Invest, 108:669-678) or the translocation domain (domain II) of Pseudomonas aeruginosa exotoxin A (ETA(dII)) (Hung, et al., (2001) Cancer Research, 61: 3698-3703) to E7 in the context of a DNA vaccine. To enhance MHC class II antigen processing, the sorting signals of the lysosome associated membrane protein (LAMP-1) have been linked to the E7 antigen, creating the Sig/E7/LAMP-1 chimera (Ji, et al, (1999), Human Gene Therapy, 10: 2727-2740).

To enhance further the potency of naked DNA vaccines, an intercellular strategy that facilitates the spread of antigen between cells can be used. This improves the potency of DNA vaccines as has been shown using herpes simplex virus (HSV-1) VP22, an HSV-1 tegument protein that has demonstrated the remarkable property of intercellular transport and is capable of distributing protein to many surrounding cells (Elliot, et al., (1997) Cell, 88: 223-233). Such enhanced intercellular spreading of linked protein, results in enhancement of HCV-specific CD8+ T cell-mediated immune responses and anti-HCV effect. Any such methods can be used to enhance DNA vaccine potency against HCV in an infected individual.

Prophylactic Vaccines Using HCV Consensus Sequences

The HCV consensus sequence taught herein can also be used as a vaccine prophylactically, i.e., lessening the symptoms associated with HCV in an individual who is infected with HCV. In one embodiment, an individual is preventing from contracting HCV. The vaccine compositions of the invention are administered to an individual who is not infected with HCV. By its ability to induce or augment an effective immune response, the vaccine mollifies the dangers of becoming infected with HCV. The individual should be given an amount effective to induce or augment effective immune responses, which can be measured using the assays detailed in the Examples section. The invention discloses epitopes which are immunogenic in individuals with certain MHC haplotypes. Thus, depending on the MHC haplotype of the individual, a customized vaccine may also be made if desired. Even if the individual does not have matching MHC haplotype, the advantage of using consensus sequence as a vaccine is that it has the potential for cross reactivity. In another embodiment, the individual has been exposed to ACV experiences a lessening of the symptoms associated with HCV. In this case, the individual is monitored by a physician or another individual suitably trained to monitor the progression of HCV infection. As disclosed above, symptoms associated with HCV include jaundice, fatigue, dark urine, abdominal pain, loss of appetite, and nausea, liver disease, and hepatocellular carcinoma. Physicians can monitor the individuals for the presence and level of HCV (e.g., by RNA levels) to assess the individual's responses to vaccination.

Methods of Treating HCV-Infected Individuals

The invention provides methods for treating individuals infected with HCV. Using the compositions comprising HCV consensus sequences disclosed herein, individuals who are infected with HCV can be treated by administering an effective amount of the composition. An effective amount is an amount that is sufficient to stimulate an immune response. The immune response can be monitored by using the assays disclosed in the Examples or any other standard measure of immune response (e.g., measuring antibodies, cytokine levels, activation markers on immune cells, etc.) Optionally, the levels of HCV can be measured by standard assays known to one of ordinary skill in the art. In one embodiment, the administration of a composition comprising HCV consensus sequences (e.g., a vaccine) will stimulate T cell and/or B cell response. The stimulation of an immune response will serve to eradicate circulating HCV in the individual.

In some embodiments, the administration of one or more HCV consensus sequence proteins or a fragment thereof is capable of inducing an immune response in a host animal. In one embodiment, the immune response is a cell-mediated immune response. In one embodiment, the effective immune response induced or augmented by the one or more HCV consensus sequence proteins or a fragment thereof comprises a T cell response, such as a CD4+ T cell response or a CD8+ T cell response, or both.

These immune cell responses can be measured by both in vitro and in vivo methods to determine if the immune response involved in the present invention is effective. Efficacy can be determined by comparing these measurements for those to whom have received treatment to those who have received no treatment. Another alternative to compare the measurements of an individual before treatment with his/her measurements after treatment. One possibility is to measure the presentation of the HCV consensus protein or consensus epitope of interest by an antigen-presenting cell. The HCV consensus protein or consensus epitope of interest may be mixed with a suitable antigen presenting cell or cell line, for example a dendritic cell, and the antigen presentation by the dendritic cell to a T cell that recognizes the protein or antigen can be measured. If the HCV consensus protein or consensus epitope of interest is being recognized by the immune system at a sufficient level, it will be processed into peptide fragments by the dendritic cells and presented in the context of MHC class I or class II to T cells. For the purpose of detecting the presented protein or antigen, a T cell clone or T cell line responsive to the particular protein or antigen may be used. The T cell may also be a T cell hybridoma, where the T cell is immortalized by fusion with a cancer cell line. Such T cell hybridomas, T cell clones, or T cell lines can comprise either CD8+ or CD4+ T cells. The dendritic cell can present to either CD8+ or CD4+ T cells, depending on the pathway by which the antigens are processed. CD8+ T cells recognize antigens in the context of MHC class I molecules while CD4+ recognize antigens in the context of MHC class II molecules. The T cell will be stimulated by the presented antigen through specific recognition by its T cell receptor, resulting in the production of certain proteins, such as IL-2, tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ), that can be quantitatively measured (for example, using an ELISA assay, ELISPOT assay, or Intracellular Cytokine Staining (ICS)).

In another aspect, the invention provides methods for preventing an already infected individual from entering a chronic phase of infection. As the infection moves into the chronic phase, an infected individual's immune system is weakened and less effective against the HCV. By utilizing the precise genetic changes disclosed herein, methods of treatment are possible to prevent the chronic phase of infection. An effective amount of a composition comprising HCV consensus sequences (e.g., a vaccine) is administered to the infected individual. The progress of the infection is monitored afterwards. Optionally, prior to the administration of a vaccine, the full or partial sequence of the HCV infecting the individual is determined and the consensus sequence for administration can be customized if desired.

Methods of Diagnosis

The invention also provides for methods of diagnosing an individual with HCV. By using the consensus sequences disclosed herein, one of skill in the art can obtain a biological sample from an individual to be tested and then use PCR primers (or any comparable molecular biological technique) to the consensus sequence to amplify nucleic acids in the biological sample to determine if the individual has been infected with a particular strain of HCV (e.g., HCV 1a or HCV 1b).

In another embodiment, the nucleic acid sequence of portions of the HCV consensus sequence can be used as probes for purposes of diagnosis. The oligonucleotide sequences selected as probes should be sufficiently long and sufficiently unambiguous that false positives are minimized. The oligonucleotide can be labeled such that it can be detected upon hybridization to the nucleic acid (e.g., DNA) being screened. Methods of labeling are well known in the art, and include the use of radiolabels, such as ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.

Kits

The invention further provides kits (or articles of manufacture) comprising the HCV consensus sequences of the present invention.

In one aspect, the invention provides a kit comprising both (a) a composition comprising a HCV consensus sequence described herein, and (b) instructions for the use of the composition in the prevention or treatment of HCV in a host. In some embodiments, the instructions are on a label. In other embodiments, the instructions are on an insert contained within the kit.

In another aspect, the invention provides a kit comprising both (a) a composition comprising a HCV consensus sequence described herein; and (b) instructions for the administration of the composition to a host. In some embodiments, the instructions are on a label. In other embodiments, the instructions are on an insert contained within the kit.

In another aspect, the invention provides a kit comprising both (a) a composition comprising a HCV consensus sequence described herein; and (b) instructions for selecting a host to which the composition is to be administered. In some embodiments, the instructions are on a label. In other embodiments, the instructions are on an insert contained within the kit.

In another aspect, the invention provides a kit comprising both (a) a composition comprising a HCV consensus sequence described herein; and (b) instructions for selecting one or more consensus sequence(s) to administer to an individual. In some embodiments, the instructions are on a label. In other embodiments, the instructions are on an insert contained within the kit

In some embodiments of each of the aforementioned aspects, the composition is a vaccine. In some embodiments of each of the aforementioned aspects, the vaccine comprises the entire consensus sequence 1a or a fragment thereof. In other embodiments, the vaccine comprises the entire consensus sequence 1b or a fragment thereof. In other embodiments, the vaccine comprises one or more HCV proteins or a fragment thereof. It is understood that these composition include non-synonymous changes as well.

The following are provided to illustrate, but not to limit, the invention.

EXAMPLES Example 1 Obtaining HCV Consensus Sequence 1a and 1b

Using HCV Sequence Database at Los Alamos National Laboratories at the website: http://hcv.lanl.gov, all available non-redundant human sequences greater than 500 nucleotides were obtained. Then, the options were set as default except for: “Genotype” was set to “1”, “Subtype” to “a or b”, “Include recombinants” set to “off”, “Include fragments longer than” was set to “on, 500” and “Exclude Non-human hosts” was set to “on.” Once the preliminary query result was displayed, “Exclude related” was chosen. Then the sequences were downloaded with default options. The rationale behind these series of steps is that size restriction reduces the number of unidirectional (poor quality) sequences.

Next, the alignments were neatened by performing any number of commands, such as restoring codon boundaries, closing gaps and removing non-translated termini. The rationale behind this step is that only the primary open reading frame is desired. Next, sequences that contain >1% ambiguous sites or >1 frameshift were removed. The rationale for this step is to remove low-quality sequences. Next, the terminal “gap” characters were converted to “missing.” The rationale behind this step is to distinguish true gaps from missing data for subsequent analysis. Using the CleanCollapse program, which was written by Stuart Ray and publicly available at <http://sray.med.som.jhmi.edu/SCRoftware/CleanCollapse>, identical sequences were identified. The CleanCollapse program has also been disclosed in Kieffer T L, et al. J Infect Dis. 2004; 189(8):1452-65. For identical sequences, related publications were checked and any linked sequences were removed. The rationale behind this approach is that oversampling individuals could skew sequence distributions. Despite use of “Exclude related” option above, the returned data set can still include many redundant/related sequences. Thus, these were removed to prevent skewing of sequence distributions.

Predicted polyprotein sequences were generated by automated translation in BioEdit (Tom Hall, URL: <http://www.mbio.ncsu.edu/RNaseP/info/programs/BIOEDIT/bioedit.html>) using standard eukaryotic genetic code. The majority-rule consensus sequence were identified for each subtype by using the MargFreq program written by Stuart Ray and publicly available at <http://sray.med.som.jhmi.edu/SCRoftware/MargFreq> to identify modal aa residue at each site. The MargFreq program has also been disclosed in Ray S C, et al. J. Virol. 1999 April; 73(4):2938-46. Where there is a near-tie at a site, the nearest neighbor (the other subtype) distribution for likely ancestor/shared state was examined and then used. The rationale behind this is that it reduces likelihood of escape mutant for common HLA allele being used. The result was consensus sequence1a (SEQ ID NO:1) and consensus sequence1b (SEQ ID NO:2).

Example 2 Cellular Immune Selection with Hepatitis C Persistence in Humans

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific cellular immune responses. To determine if immunologically-driven sequence variation occurs with HCV persistence, we coordinately analyzed sequence evolution and CD8+ T cell responses to epitopes covering the entire HCV polyprotein in subjects followed prospectively from prior to infection to beyond the first year. There were no substitutions in T-cell epitopes for a year following infection in a subject who cleared viremia. In contrast, in subjects with persistent viremia and detectable T cell responses, we observed substitutions in 69% of T cell epitopes, and every subject had a substitution in at least one epitope. In addition, amino acid substitutions occurred 13-fold more often within than outside T cell epitopes (p<0.001, range 5-38). T lymphocyte recognition of eight of ten mutant peptides was markedly reduced compared to the initial sequence, indicating viral escape. Of 16 non-envelope substitutions that occurred outside of known T cell epitopes, eight represented conversion to consensus (p=0.015). These findings reveal two distinct mechanisms of sequence evolution involved in HCV persistence: viral escape from CD8+ T cell responses and optimization of replicative capacity.

The World Health Organization estimates there are 170 million persons with HCV infection worldwide, and an estimated 4 million persons are infected with hepatitis C virus (HCV) in the United States.(1,2) In most countries, HCV infection is found in 1-2% of the general population and may cause cirrhosis or hepatocellular cancer, but only when infection persists.(3-7)

Patients in the acute phase of HCV infection are much more likely to respond to therapy designed to eradicate the virus than are patients after progression to chronicity.(8-10) The features unique to acute infection that allow increased responsiveness to interferon therapy remain unknown. Spontaneous clearance of HCV infection occurs in about 20% of acutely infected individuals and is associated with a broadly specific and vigorous cellular immune response.(11-14) Although the cellular immune response is often less vigorous and more narrowly directed in those who fail to clear the infection, a cellular immune response is nonetheless often present in early infection and may even persist into chronic infection. Why the early response fails to control viremia in those who progress to chronic infection is not clear, but responses generated in acute infection have been shown to decline in some subjects who remained persistently infected and chronic infection is characterized by low frequencies of CD8+ T cells in peripheral blood.(13, 15-20) Although the liver has the potential to delete antigen-specific T cell responses, HCV-specific CD8+ CTL lines have been generated from the liver of both chronically infected humans and chimpanzees, suggesting that elimination of HCV specific lymphocytes from the liver is neither universal nor necessary for HCV persistence.(21-24) Survival of HCV despite virus-specific CD8+ CTL might be explained by impaired cellular effector functions (proliferation, cytokine secretion, or cytolytic activity), T cell exhaustion, or dendritic cell dysfunction.(16, 25-27) A final possibility is that persistence is facilitated by viral evolution over the course of infection, enabling escape by mutation of key epitopes targeted by T-lymphocytes. Mutational escape from T cell responses has indeed been noted in HIV, which uses an error prone RNA polymerase similar to HCV.(28)

Mathematical models of viral kinetics suggest that up to 10¹² virions are produced each day in a human with chronic hepatitis C.(29) This rate exceeds comparable estimates of the production of HIV by more than an order of magnitude, and, coupled with the absence of proofreading by the HCV NS5B RNA polymerase, results in frequent mutations within the HCV genome. Mutation of class I or II MHC restricted T cell epitopes could alter the outcome of infection by preventing or delaying clearance of infected hepatocytes.(30) In the face of a vigorous multispecific CTL response, mutation of several epitopes, perhaps simultaneously, would be required for survival of the virus. In HCV infection, a strong association between viral persistence and the development of escape mutations has been demonstrated in the chimpanzee model (31) and one group examined viral evolution in a single human histocompatibility leukocyte antigen (HLA)-B8-restricted NS3 epitope,(32) but evidence of evasion of a multispecific CTL response in humans is lacking. Although mutations in class I MHC restricted HCV epitopes have been observed in humans with chronic HCV infection, it is uncertain that these mutations result from CD8+ T cell selection pressure or that they occur during the acute phase of infection, when clearance or persistence is determined.(33-35)

Studies of the cellular immune response to acute HCV infection have been challenging because acute hepatitis C is usually clinically silent, making early virus isolates and CTL difficult to obtain. In addition, no consistent pattern of HCV epitope dominance has emerged in humans so large numbers of PBMC are needed for the broad screening required to identify CTL responses. We have overcome these challenges to test the hypothesis that cytotoxic T lymphocyte (CTL)-driven sequence variation occurs with progression to persistent HCV in humans. We prospectively studied HCV antibody negative injection drug users at risk for HCV infection and compared the viral sequences at initial viremia to sequences obtained at multiple time points in acute HCV infection. In parallel, a genome wide analysis of T cell responsiveness was performed. As evidence of immune selection pressure, we determined the percentage of T cell epitopes that underwent substitution, assessed the likelihood of amino acid changing substitutions to occur within versus outside T cell epitopes, and examined the effects of observed amino acid sequence changes in epitopes on T cell recognition and MHC class I binding. For amino acid substitutions outside T cell epitopes, we investigated explanations other than T cell pressure. Our results provide strong evidence in humans that both immune and fitness selection occur during the acute phase of HCV infection.

Materials and Methods

Participants. The Risk Evaluation Assessment of Community Health (REACH) prospective study of young IDUs in Baltimore, Md. examined the incidence and risk factors for HCV infection, as described previously. (47) Participants eligible for the study were anti-HCV antibody negative, between 15 to 30 years of age, and acknowledged use of injection drugs. Participants were invited to co-enroll in a substudy of acute hepatitis C and those who consented had blood drawn for isolation of serum, plasma, and peripheral blood mononuclear cells in a protocol designed for monthly follow up. At each visit, participants were provided counseling to reduce the risks of drug use. The REACH protocol and the HCV substudy protocols were approved by the institutional review boards of the Johns Hopkins Schools of Medicine and Hygiene and Public Health.

From 1997 to 2002, 179 participants were enrolled and 62 (34.6%) developed anti-HCV antibodies (seroconverted). Beginning in November 2001, acutely infected persons were assessed for donation of ˜10⁸-10¹⁰ PBMC by blood donation or apheresis. HCV-specific lymphocyte responses and sequences were studied in detail in 5 acutely infected persons who were assessed frequently during the six-month period following infection and from whom large volumes of PBMC were obtained. HCV sequencing and limited analysis of the cellular response were performed using an additional 3 subjects for whom smaller numbers of cells were available, as described below.

HCV testing protocol. Serum or plasma, stored at −80° C., from monthly follow-up visits was tested for the presence of HCV specific antibodies using the commercially available Ortho version 3.0 enzyme-linked immunosorbent assay (ELISA) (Ortho Clinical Diagnostics, Raritan, N.J.) according to manufacturer's instructions. Specimens in which antibodies were detected were retested in duplicate along with the participant's previous seronegative sample to identify seroconverters. HCV RNA testing was performed on sera or plasma separated from blood within two hours of collection and stored at −80° C. For HCV seroconverters, HCV RNA testing was done on samples collected before seroconversion until a negative result was obtained and after seroconversion to evaluate the outcome of infection (viral persistence versus clearance) by using quantitative, and if undetectable, qualitative HCV RNA tests that are described below.

HCV RNA Assays—Qualitative. For detecting HCV RNA, we used the COBAS AMPLICOR™ Hepatitis C Virus Test version 2.0 (Roche Molecular Systems, Branchburg, N.J.). A limit of detection of 1.7 log₁₀International Units (IU)/mL at >95% detection is reported for this assay. Quantitative. To determine concentration of HCV RNA in serum, we used a quantitative RT-PCR assay (COBAS AMPLICOR™ HCV Monitor version 2.0, Roche Molecular Systems). This assay has a lower limit of quantitation of 2.8 log₁₀ IU/mL. When HCV RNA was not detected by using this assay, the sample was retested using the Roche qualitative test.

HCV Genotyping. Genotype was determined by performing phylogenetic analysis on Core-E1 region sequences of HCV obtained from the first viremic specimen. For most specimens, sequences were obtained from cDNA clones generated with a long amplicon RT-PCR method that has been described previously.(48) For other specimens, genotype was determined by direct sequencing of RT-PCR products from the same Core-E1 region as previously described.(49) Sequences were aligned using ClustalX,(50) trimmed to equal length using BioEdit.(51) The GTR+I+G analytical model (parameters available on request from the authors) was selected using the AIC criterion as implemented in ModelTest version 3.06(52) and PAUP* version 4b10 (Sinauer Associates, Sunderland, Mass.). Phylogenetic trees were estimated using the neighbor-joining algorithm implemented in PAUP*, and robustness of clustering was tested using by bootstrap analysis.(53)

Viral recovery. HCV clearance was defined as the presence of anti-HCV with HCV RNA undetectable by the COBAS AMPLICOR™ qualitative assay in serum or plasma specimens from ≧2 consecutive visits obtained at least 300 days after initial detection of viremia. Persistence was defined as the persistent presence of anti-HCV with HCV RNA detectable by the qualitative or quantitative COBAS AMPLICOR assay in serum or plasma specimens obtained at least 300 days after initial viremia.(54)

Hemigenomic HCV sequencing and analysis. From 140-280 uL of serum or plasma, the 5.2 kb region from the 5′UTR to the NS3/NS4A junction was cloned as previously described.(48) For each specimen, thirty-three clones were assigned to clonotypes by using a previously-described gel shift assay,(55) and 2 clones representing the modal clonotype were sequenced, with a third clone used as needed to resolve discrepancies. Sequences were assembled into contigs using Aligner (CodonCode). Sequence data were obtained at the point of initial viremia and approximately six months later.

Reference sequence analysis. Reference sequence data were obtained from the HCV Sequence Database (http://hcv.lanl.gov). For table 1, amino acid sequence was inferred from cDNA sequences, which were obtained using the HCV Sequence Database's Search Interface. Default search parameters were used, except that (i) only subtype 1a sequences were included, (ii) recombinant sequences were excluded, (iii) non-human sequences were excluded, and (iv) the search was performed for the codon of interest based on position in the HCV polyprotein. When multiple sequences with the same “patient ID” were obtained, only the first occurrence was retained.

Cellular immunology. IFN-γ ELISPOT assay to assess HCV-specific T-cell responses. HCV-specific CD8+ T-cell responses were quantified by ELISPOT assay as previously described(56) with the following modifications. For Subjects 17, 18, 21, 28, and 29, enough PBMC were acquired to test for T cell recognition of 523 overlapping peptides (16-22mer peptides overlapping by 10 amino acids) spanning the entire expressed HCV-H77 genome (genotype 1a) as well as 83 peptides corresponding to optimal described CTL epitopes(57) in a matrix format. The peptides recognized in the matrix were subsequently tested individually and in at least duplicate to confirm recognition and to measure the number of spot forming colonies (SFC) produced. For three additional subjects where the number of cells was limited, responses to peptides spanning regions of sequence variation detected during sequencing, but not to the entire collection of 523 overlapping peptides, were assessed. Ninety-six well polyvinylidene plates (Millipore, Billerica, Mass.) were coated with 2.5 μg/ml recombinant human anti-IFN-gamma antibody (Endogen, Pierce Biotechnology, Rockford, Ill.) in PBS at 4° C. overnight. Fresh or previously frozen PBMC were added at 200,000 cells/well in 140 μl R10 media (RPMI 1640 (Sigma-Aldrich Corp., St. Louis, Mo.), 10% FCS (Sigma-Aldrich), and 10 mM Hepes buffer(Sigma-Aldrich) with 2 mM glutamine and antibiotics (50 U/ml penicillin-streptomycin)). Peptides were added directly to the wells at a final concentration of 10 μg/ml. The plates were incubated for 20 hours at 37° C., 5% CO₂. Plates were then washed, labelled with 0.25 μg/ml biotin-labelled anti-IFN-γ (Endogen), and developed by incubation with streptavidin-alkaline phophatase (Bio-Rad Lab., Hercules, Calif.) followed by incubation with BCIP/NBT (Bio-Rad) in Tris-buffer (pH 9.5). The reaction was stopped by washing with tap water and the plates were dried, prior to counting on an ELISPOT reader (Cellular Technology Ltd, Cleveland, Ohio). For quantitation of ex-vivo responses, the assay was performed at least in duplicate and background was not more than 15 SFC per million PBMC. Responses were considered positive if the number of spots per well minus the background was at least 25 SFC per million PBMC.(56) A control of pooled cytomegalovirus, Epstein-Barr virus, and influenza antigens (CEF control peptide pool) and phytohemmaglutinin (PHA) were used as positive controls.(58) Responses to the CEF control peptide pool were quantifiable and remained relatively constant over time. Responses to PHA were uniformly positive.

Intracellular cytokine staining (ICS). To determine whether the lines generated for each epitope were CD4+ or CD8+ T cell lines, intracellular cytokine staining (ICS) for IFN-γ was performed as previously described.(56) Briefly, 1×10⁶ PBMC were incubated with 4 μg/ml peptide at 37° C. and 5% CO₂ for 1 h before the addition of Monensin (1 μl/ml; Sigma-Aldrich). The cells were incubated for an additional 5 h at 37° C. and 5% CO2. PBMC were then washed and stained with surface antibodies, fluorescein isothiocyanate (FITC)-conjugated anti-CD8 or FITC-conjugated anti-CD4 (Becton Dickinson, BD, Franklin Lakes, N.J.) at 4° C. for 20 min. Following the washing, the PBMC were fixed and permeabilized (Caltag, Burlingame, Calif.), and the phycoerythrin (PE)-conjugated anti-IFN-γ MAb (Becton Dickinson) was added. Cells were then washed and analyzed on a FACS-Calibur flow cytometer using CellQuest software (Becton Dickinson).

Magnetic bead separation of CD8+ and CD4+ T cells. To determine if the responding T cells in PBMC were CD4+ or CD8+ T cells, between 3 and 10×10⁷ PBMC were labeled with magnetic beads bearing anti-CD8 or anti-CD4 antibodies (Miltenyi Biotec, Auburn, Calif.) according to the manufacturer's instructions and cells were positively selected using an Auto MACS (Miltenyi Biotec) to isolate either CD8+ or CD4+ cells. The ELISPOT assay was repeated using the isolated CD8+ or CD4+ T cells to determine if recognition of an epitope were mediated by CD8+ or CD4+ T cells.

Bulk stimulation of peripheral blood mononuclear cells. To establish CD8+ T cell lines, cryopreserved or fresh PBMC (4−10×10⁶) were stimulated with 10 μg/ml of synthetic HCV peptide and 0.5 μg/ml of the costimulatory antibodies anti-CD28 and anti-CD49d (Becton Dickinson) in R10 media. Recombinant interleukin-2 (IL-2, 25 IU/ml) was added on day 2 and every other day thereafter. Cells were restimulated with 25×10⁶ irradiated allogeneic PBMC and 10 μg/ml of synthetic HCV peptide after ten days.

Testing impact of amino acid substitutions on T cell recognition. To assess the impact of amino acid substitutions on T cell recognition, HCV peptide-specific T cell lines generated from PBMC obtained six months after initial viremia were tested for IFN-γ production in response to serial dilutions of synthetic peptides representing the viral sequences present at initial viremia (t₀) or at six months following initial viremia (t₆). In five cases where the frequency of T cells specific for the HCV epitope was high, bulk PBMC obtained six months following initial viremia were also tested in this way. Comparison of initial and variant epitopes was performed using log₁₀ dilutions of the t₀ and t₆ peptides from 10 μM to 0.001 μM in the IFN-γ ELSIPOT assay described above for PBMC, but using 30,000 T cells when T cell lines were tested. Ten peptide pairs from subjects with chronic viremia were tested, and three patterns were observed. Loss of recognition was defined as either no recognition at the highest concentration of the t₆ peptide or at least 20 fold greater recognition of the to peptide than the t₆ peptide at all concentrations. Decreased recognition was defined as greater than 2 and less than 20 fold fewer spot forming colonies (SFC) produced at two or more concentrations of the t₆ peptide. Comparable recognition was defined as no more than a two fold difference in SFC between the t₀ and t₆ peptides at two or more concentrations tested.

MHC-peptide binding assays. EBV transformed cell lines were used as the primary sources of HLA molecules. Cells were maintained in vitro and HLA molecules purified by affinity chromatography as previously described.(59) Quantitative assays to measure the binding of peptides to purified class I molecules are based on the inhibition of binding of a radiolabeled standard peptide.(59) Briefly, 1-10 nM of radiolabeled peptide was co-incubated at room temperature with 1 μM to 1 nM of purified MHC in the presence of 1 μM human, β2-microglubulin (Scripps Laboratories, San Diego, Calif.) and a cocktail of protease inhibitors. After a two-day incubation, binding of the radiolabeled peptide to the corresponding MHC class I molecule was determined by capturing MHC/peptide complexes on Greiner Lumitrac 600 microplates (Greiner Bio-one, Longwood, Fla.) coated with the W6/32 antibody, and measuring bound cpm using the TopCount microscintillation counter (Packard Instrument Co. Meriden, Conn.).

Statistical analysis. Statistical analysis was done with the aid of SigmaStat software version 3.10 (Systat software, Inc.). For comparing proportions, Fisher's exact (small sample size) and Chi squared (large sample size) tests were used. Differences were considered significant if p-values were <0.05.

Results—We assessed T cell responses and sequenced half the HCV genome in eight subjects, seven of whom progressed to chronic infection (FIG. 1). T cell and viral analyses were done for all eight at initial detection of viremia and then six months later. Additional assessment of T cell responses was done at the time points designated by arrows in FIG. 1. No T cell responses were detectable at initial viremia, but all of the subsequently detected T cell responses were present by 6 months following infection.

Persistence versus Loss of T cell Epitopes with Sequence Evolution—The locations of amino acid substitutions and recognized CD8+ T cell epitopes are shown for subjects 17, 18, 21, 28, and 29 in FIG. 2. Subject 21 was the only subject with no detectable T cells responses. The only subject who cleared HCV spontaneously (18) was also the only individual whose HCV genome had no substitutions within any recognized T cell epitope at 6 or 12 months following initial viremia. Subjects 17, 28, and 29 remain persistently infected and had substitutions at six months in ⅗, 6/8, and ⅔ of recognized CD8+ T cell epitopes, respectively. In summary, the subject who cleared infection had no substitutions in 3 recognized T-cell epitopes at 6 or at 12 months after infection, whereas the three subjects with chronic viremia and T cell responses had substitutions in 60-75% of CD8+ T cell epitopes by six months following infection.

Impact of Amino Acid Substitutions on T cell Recognition—T cell lines were generated from PBMC using synthetic peptides representing the viral sequences present at initial viremia (t₀). To assess the impact of amino acid substitutions on T cell recognition, those T cell lines and bulk PBMC obtained approximately 6 months after initial viremia were tested for IFN-γ production in response to serial dilutions of the to peptide or a synthetic peptide representing the viral sequence present at six months following initial viremia (t₆). Ten t₀/t₆ peptide pairs from subjects with persistent viremia were tested using T cell lines, and three patterns were observed (FIG. 3). For the ten t₆ peptides tested, we noted loss of recognition of four (FIG. 3 a), decreased recognition of four others, (FIG. 3 b), and comparable recognition of two (FIG. 3 c). Therefore, for 8 of the 10 mutations in recognized epitopes tested, recognition by T lymphocytes was lost or significantly reduced compared to recognition of the sequence present at initial viremia, indicating escape. In no case was the t₆ variant peptide recognized better than the to peptide. For five t₀/t₆ peptide pairs also tested with bulk PBMC, the patterns of recognition were the same as those observed using T cell lines, as shown for one peptide pair in FIG. 4. That PBMC as well as T-cell lines generated against the to peptides failed to recognize the t₆ peptides suggests that not only did the substitution allow escape from the T-cells specific for the original sequence, but also that no new T-cell responses were generated against the t₆ sequence. To rule out transient suppression or problems with specimen handling and the subsequent development of T cell responses to the t₆ HCV sequence, IFN-γ ELISPOT testing for recognition of the t₆ peptides demonstrating escape was also performed in subjects 17 and 28 using PBMC obtained approximately 12, 18, 24, and in subject 17, 36 months following initial infection. The patterns of recognition persisted over time and recognition of the t₆ peptides declined in parallel with the decline in recognition of the to peptides that occurred with prolonged infection (FIG. 5). Despite months of persistent exposure to the t₆ peptides that escaped recognition at six months following infection, in no case did a new T cell response specific for a t₆ peptide arise in the following 6 to 36 months.

Mechanisms of Loss of T cell Recognition with Amino Acid Substitutions—Amino acid substitutions may result in decreased recognition through reduced HLA binding capacity, abrogation of T cell recognition, or altered processing with failure to generate the correct sequence for presentation on the surface. We did observe marked reduction in HLA biding capacity as one mechanism for reduced recognition in our subjects. For example, the HLA A*0101-restricted ATDALMTGY epitope recognized at to by Subject 28 had an A*0101 binding capacity (IC₅₀) of 0.24 nM while the ATDALMTGF peptide recognized at t₆ had a binding capacity of 64 nM. A five fold difference in binding capacity is considered significant, thus the variant peptide is less well bound to the HLA. However, the HLA A*0201-restricted KLVALGINAV epitope recognized at to by Subject 28 had an A0201 binding capacity of 5.0 nM while the KLVAMGINAV peptide recognized at t₆ had a binding capacity of 2.3 nM, an insignificant difference that if anything favors the less well recognized peptide, t₆. This t₆ peptide may stimulate less IFN-γ production in the ELISPOT assay because of decreased T cell recognition rather than reduced HLA binding capacity. Although two of the ten t₀/t₆ peptide pairs were recognized comparably, we can not rule out that they represent escape mutations as well since substitutions have been shown by others to result in altered processing such that the peptide is no longer presented on the cell surface.(32,36,37) This mode of escape is circumvented when peptides are loaded onto the surface of the cell, as is the case in an ELISPOT assay.

Driving Forces for Sequence Evolution—We next evaluated the proportion of substitutions occurring in observed T cell epitopes and investigated explanations for substitutions outside of T cell epitopes. Of 69 substitution observed in the eight subjects, 17 (25%) occurred within detected CD8+ T-cell epitopes, 36 (52%) in envelope proteins (likely antibody targets), and 16 (23%) outside envelope regions and T cell epitopes. Of the eight subjects, five subjects had persistent viremia and T cell responses, and amino acid substitutions were a median of 13 fold more likely to occur within T cell epitopes than outside epitopes (p<0.001, range 5-38) in those subjects. Even though at a population level the envelope proteins are highly diverse, amino acid substitutions in T cell epitopes exceeded those in E1 and E2 by 7 fold (p<0.001, range 4-14). Of the 16 amino acid substitutions observed outside of the envelope proteins or recognized T cell epitopes (Table 1), 8 represented conversion to the most commonly-observed residue in reference sequences (i.e., convergence to the consensus sequence). Because each change has 19 alternative amino acids, and each site has only one modal (most frequently-observed) state, the convergence on modal residues at 50% of sites is highly unexpected if substitution is random (p=0.015, Table 2). In addition, the convergent changes were not consistently conservative changes, arguing against strict structural or functional constraint. These data are consistent with an accompanying study of chronically infected individuals for whom the inoculum sequence was known. That study showed that substitutions occurring in known epitopes for which the infected individual lacked the presenting HLA allele were consistently reversions toward the worldwide consensus sequence for subtype 1b.

Discussion—In this investigation of sequence variation in T cell epitopes as a potential mechanism for viral persistence, we show that amino acid substitutions during acute HCV infection are nonrandom and may be explained in part by escape from CD8+ T cell recognition and convergence, possibly because of replicatively unfit substitutions selected in a previous host. Significantly, there is early fixation of the T cell repertoire since we observed no instances of de novo development of T cells that recognized mutant t6 epitopes better than the original t0 epitope using lines or PBMC.

Using the chimpanzee model of HCV infection, mutation of multiple class I MHC restricted epitopes early in the course of chronic HCV infection has been demonstrated.(31) The role of CD8+ CTL in control of HCV replication was further reinforced by a statistically significant increase in the number of mutations resulting in amino acid changes in class I MHC restricted epitopes but not unrestricted epitopes or flanking sequences of the viral genome. These data indicate that in chimpanzees, amino acid substitutions in class I MHC restricted epitopes are selected and possibly maintained by HCV-specific CD8+ CTL populations that exert positive Darwinian selection pressure.

We also observed a statistically significant increase in the number of mutations resulting in amino acid changes in class I MHC restricted epitopes versus portions of the viral genome outside T cell epitopes and that amino acid substitutions in class I MHC restricted epitopes resulted in escape from CD8+ T cell recognition in acutely HCV-infected humans who progressed to chronic infection. New T cells specific for the sequences detected six months after initial viremia were not detected despite follow-up for as long as three years following infection. Thus, selection of HCV variants that evade CD8+ T cell recognition may represent a mechanism for persistence of HCV infection in humans. Supporting this, we observed no substitutions within recognized CD8+ T cell epitopes in the subject who cleared infection and substitution in 60-75% of CD8+ T cell epitopes in subjects with persistent infection. The number of CTL epitopes with substitutions has previously been shown to correlate with control of HIV viremia,(28) but a relationship between HCV control and maintenance of T cell epitopes had not been shown previously.

Although the observed substitutions were disproportionately contained within the portion of the HCV polyprotein in T-cell epitopes, many were found outside detectable T cell epitopes. It is possible that we missed CD8+ T cell epitopes and that some of the substitutions observed outside of T cell epitopes actually represented substitutions within T cell epitopes. We took several steps to minimize the chances of this occurring. Where there were substitutions and the H77 sequence used to make overlapping peptides as potential antigens differed from that of the subject, we tested for recognition of overlapping peptides representing autologous sequence. Where the subject's sequence matched H77 in areas of amino acid substitution, but there were no responses detected, we tested for recognition of additional overlapping peptides with different termini to minimize the possibility that we cut within a region containing an epitope. We detected no additional epitopes via ELISPOT analysis with either method of antigen modification (data not shown).

Since there is no clinical indication for liver biopsy in acute infection, we could only assess responses in the periphery. It is therefore possible that some of the substitutions may represent pressure for T cell responses present in the liver but not detectable in the periphery, We do not favor this explanation since previous studies have shown that the majority of T cell responses in the liver are also detectable in the peripheral blood.(38,39) There are several possible alternative mechanisms for selection of these substitutions occurring outside of observed CD8+ T cell epitopes. The first is that they represent substitutions in CD4+ T cell epitopes. Although we detect a few CD4+ T cell epitopes using our ELISPOT assay, the conditions of the assay preferentially detect CD8+ T cell epitopes and we may fail to detect all the possible CD4+ T cell epitopes. The second possible explanation is that they represent substitutions in B cell epitopes. Mutations in dominant antibody epitope(s) located in the hypervariable region 1 (HVR-1) of envelope glycoprotein 2 (E2) have been linked to persistence of HCV infection.(40) B cell epitopes are predicted to occur within the envelope regions of the polyprotein and the majority of mutations outside T cell epitopes were found in the envelope proteins for most subjects. Another possible explanation is that the mutations outside of T cell epitopes are selected because they confer a viral replication advantage. Some mutations may represent epitopes recognized by the previous host that revert to a more stable sequence when the new host fails to mount similar immune pressure. This may occur when the new host lacks the MHC allele required to present that epitope. Loss of escape mutations upon passage of SIV to new animals (41), and HIV to humans (42), that do not exert immune pressure on that region has been described recently, with the inference that escape from CTL responses may reduce viral fitness.

The relevance of those studies to human infection with HCV is supported by a recent study of one epitope in an acutely HCV infected patient (32), and our accompanying study of chronically infected individuals. The latter study shows that HCV amino acid sequence tends to revert to consensus in areas outside of T cell epitopes in subjects who are persistently infected. The consensus sequence likely represents a more replicatively fit state than the initial infecting strains, which have presumably adapted to the immune response of the previous host. Taken together, these results reveal at least two types of sequence variation occurring simultaneously in progression of acute HCV to persistence: immune pressure that selects T cell escape variants, and reversion to consensus sequence that is likely to result in enhanced replicative fitness.

Despite viral mutation resulting in the production of new potential antigens, no new T cell responses developed in response to mutant peptides that escaped initial recognition over months and in some cases years following the appearance of the mutation. This phenomenon has also been observed with HIV sequence evolution and there the failure to prime new responses may be due to impaired CD4+ T cell function. Although overall CD4+ T cell function is intact in HCV infection, chronic HCV infection has been linked to loss of HCV specific CD4+ T cell responses.(43) In addition, HCV has been linked to impaired DC function, decreased IRF3 signaling, and PKR inhibition, which may inhibit priming of an immune response to the mutated peptides.(26,27,44,45) However, the failure to prime responses to the newly generated sequences is observed even in HIV infected individuals with relatively high CD4 counts and there is no evidence in those with HCV infection of impaired priming of immune responses to other antigens, as would be evident by global immunosuppression. An alternative explanation is that original antigenic sin (the higher threshold required for stimulation of an immune response to an epitope resembling a previously recognized epitope (46)) may be responsible for the lack of response to mutant sequences seen in HIV and HCV, though this phenomenon has not been demonstrated in humans. Lastly, since the selective pressures of the immune system favor the emergence of a viral sequence that fails to elicit a productive response, we may be observing sequences that cannot be processed effectively for presentation or that resemble self antigens and are therefore incapable of stimulating an immune response.

Although the features of acute infection responsible for increased responsiveness to interferon therapy are unknown, this study suggests a mechanistic linkage between viral sequence variation and progression to chronicity. The arrested development of new T cell responses despite ongoing viremia with sequence evolution distinguishes the acute and chronic phases of HCV infection. Enhanced understanding of cellular immune failure leading to chronic HCV infection could accelerate development of vaccines to prevent the development of chronic infection, and agents that could increase responsiveness to interferon-based therapy for chronic HCV infection.

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Example 3 Divergent and Convergent Evolution Following a Common-Source Outbreak of Hepatitis C

The genomic sequences of viruses that are highly mutable and cause chronic infection tend to diverge over time. We report that these changes represent both immune driven selection and, in the absence of immune pressure, reversion toward a more “fit” ancestral consensus. Sequence changes in hepatitis C virus (HCV) structural and nonstructural genes were studied in a cohort of women accidentally infected with HCV in a rare common-source outbreak. We compared sequences present in serum obtained 18-22 years after infection to sequences present in the shared inoculum and found that HCV had evolved along a distinct path in each woman. Amino acid substitutions in known epitopes were directed away from consensus in persons having the HLA allele associated with that epitope (immune selection), and toward consensus in those lacking the allele (reversion). Vaccines for genetically-diverse viruses may be more effective if they represent consensus sequence, rather than a human isolate.

A virus capable of genetic variation and of causing chronic infection will evolve to optimize its fitness in each host, which is the net sum of immune recognition (positive selection) and functional constraint on replication (negative selection). Because an estimated 10¹² virions are produced each day through an error-prone, non-proofreading NS5B RNA polymerase, hepatitis C virus (HCV) is especially capable of viral evolution (1,2) (These numbers in Example 3 refer to the list of references listed after Example 3). However, we previously showed that evolution is not driven by replication alone. In the acute phase of infection before adaptive immune responses (but after weeks of replication supporting a viral RNA level of more than 105 IU/mL), the same major viral variant was detected in each of a serial passage of eight chimpanzees (3). In contrast, the sequence of envelope genes, particularly HVR1, changes in virtually all humans who have been persistently infected (including the source of the inoculum passaged through this chimpanzee lineage (4)), a notable exception being persons with agammaglobulinemia, who have been shown to have reduced variability in HVR1 (5). Longitudinal studies of chimpanzees experimentally infected with HCV have revealed that amino acid replacements in immunodominant CD8+ T cell epitopes presented on MHC class I in an allele-restricted manner contribute to viral persistence (6). Thus, we hypothesized that the net evolution of HCV would demonstrate functional constraint (reversion of sequences toward consensus) as well as positive pressure (and thus reveal immunodominant epitopes).

Although it required that persons be infected with the same inoculum, it was possible to test this hypothesis because between May 1977 and November 1978 over 500 women were inadvertently infected with HCV from a single acutely-infected source, as a result of treatment with contaminated anti-D immune globulin (7). In a single amplicon, a 5.2 kb cDNA spanning 5′UTR through the NS3/NS4A junction was cloned from serum collected from 22 women 18-23 years after infection, as well as in 2 specimens of frozen plasma from the inoculum donor.

Methods and Materials

Study subjects. Twenty-two women from this outbreak were studied because they provided consent and had at least one of the 3 most common alleles at the HLA A locus (A*01, A*02, or A*03) (16).

Hemigenomic cDNA cloning. The region encoding Core, E1, E2, p7, NS2, and NS3 was amplified and cloned as previously described (17), and 40 clones per specimen were stored. For each specimen, envelope sequences from 10 random randomly-selected clones were determined using primer H77-1868a21 (17) on a PRISM version 3100 sequencer (ABI, Foster City, Calif.).

Estimation of Consensus Sequence. An alignment of full-length HCV subtype 1b sequences was obtained from the Los Alamos National Laboratories HCV database (http://hcv.lanl.gov). The alignment was edited by hand to remove gaps introduced for alignment to other genotypes, and to remove duplicate sequences from the same human source and those obtained from non-human sources. The resulting alignment included 83 sequences. A majority-rule consensus sequence was formed, with residues occurring in less than 42 sequences flagged as non-consensus. Changes in the anti-D recipients were then classified as “toward” (change results in a residue matching the consensus) or “away” (change results in a residue not matching the consensus, or residue is non-consensus).

Estimation of the likelihood of convergence. The expected frequency of co-variation assuming independence was calculated as the product of the marginal frequencies, and compared to the observed value using the Chi-squared distribution with 3 degrees of freedom. If we assume that all amino acid replacements are equally likely over a time period that is very long with respect to the rate of mutation, then sharing of amino acids at 4 variable sites in just 2 study subjects would be expected to occur at a frequency of 1/204 or 0.00000625. Of course, all amino acid replacements are not equally likely, even in the highly variable HVR1 (8); therefore, the likelihood of sharing of 4 variable sites by 2 subjects would be higher, e.g. 1/34 or 0.012 if each site is equally likely to have one of 3 residues. Because the likelihood of shared residues at variable sites in multiple study subjects is the product of such probabilities, the observed findings in this study are clearly incompatible with random substitution and most consistent with convergent evolution.

Phylogenetic Analysis. Sequences were aligned using ClustalX (18), codon boundaries were restored by hand in BioEdit (19), and phylogenetic analysis was performed using PAUP* version 4b10 (Sinauer Associates, Sunderland, Mass.) using a HKY85+G model and parameters (Ti/Tv 2.78, gamma=0.37) selected with the aid of ModelTest (20). Initial results from one specimen were consistent with subtype 1a, and that specimen was not examined further. Reference sequences included 3 from subtype 1a, 83 from subtype 1b (including AF313916), 2 from subtype 1c, 6 from subtype 2a, 8 from subtype 2b, 1 each from subtypes 2c and 2k, 4 from subtype 3a, and 1 each from subtypes 3b, 3k, 4a, 5a, 6a, 6b, 6d, 6g, 6h, and 6k (obtained from http://hcv.lanl.gov). VarPlot was used to calculate nonsynonymous and synonymous distances using the method of Nei and Gojobori, in a sliding window 20 codons wide, moving in 1 codon steps, as previously described (21).

Sequence logos. The sequence logos in FIG. 7 were generated using a novel software program, V is SPA (Visual Sequence Pattern Analysis, available on request from the author S. C. R.). The algorithm is identical to that described for type 2 logos by Gorodkin et al (22), except that the a priori distributions for the logo are calculated empirically from input sequences, and missing values in the a priori distribution are assigned the lowest frequency of residues at that site (if more than one state is represented) or 1/20 if the a priori distribution has only one residue at that site.

Results and Discussion

HCV envelope sequence from the inoculum clustered near the base of the clade formed by sequences from the chronically-infected women (FIG. 6), and the entire anti-D cohort clade was clearly distinct from all other sequences in available databases (excluding those from this outbreak), consistent with the previously-reported clinical history of common-source infection from an acutely infected donor (7). Nonetheless, HCV sequences in each woman diverged along distinct paths.

There was strong evidence of negative selection in both the regional differences in genetic divergence, as well as by comparison of the rates of nonsynonymous (amino acid-changing) and synonymous (silent) change (FIG. 7). Overall, the highest rate of non-synonymous change was observed in the E2 gene, followed by NS2, p7, E1, NS3, and Core. Synonymous substitution rates were consistently higher than nonsynonymous rates for all genes, suggesting strong negative selection is a consistent feature of chronic HCV infection. HVR1 sequences were highly divergent at many sites, but constrained at others (8,9).

The HVR1 sequences illustrated two distinct patterns of sequence change. When the amino acid of the inoculum matched the consensus for that viral subtype, residues either did not change or changed in an apparently ‘sporadic’ fashion (risk of finding the residue in recipients was not different than finding the residue in the inoculum, indicated by near-zero height of the sequence logo in FIG. 7. In contrast, when the amino acid in the inoculum differed from the consensus, there was convergent evolution toward consensus (residue was found >2 times more often in the recipients than in the inoculum, FIG. 7). For example, 16 of the 22 women had replaced H in the inoculum with R at position 394, 12 replaced A with T at 396, 11 replaced L with F at 399, and 16 replaced T with S at 401. Four women had all 4 of these changes, not significantly different from the expected frequency of 3.2, indicating that these changes occurred independently. Since there is an infinitesimal likelihood that these same amino acid substitutions occurred by chance in each of the women, these data indicate that the sequences converged to a more fit state, implying that prior evolution of the inoculum sequence to optimize its fitness in the original host resulted in changes that diminished its fitness in the subsequent hosts.

Rather than convergent evolution, these results might have been due to shared selection and then divergence (at other sites) from a rare variant that we did not detect in the inoculum. We did detect one clone (clone #5) among 20 in the inoculum material that carried the RxTxxFxS motif at positions 394-401, but it was highly divergent from all other sequences described here, as; evident from its position in the phylogenetic tree (FIG. 6B), and therefore less likely than the other 19 sequences to represent the founder strain for these women. While it is possible that a less divergent RxTxxFxS clone was present in the inoculum at very low frequency, shared selection of such a rare variant would support the same conclusion.

Because HVR1 is a potential target of both humoral and cellular immunity and the precise recognition motifs remain difficult to identify due to the extreme variability, further examination of immune selection and convergence was focused on other genes, and in particular, on known MHC class I-restricted epitopes. Consistent with immune selection hypothesis, the number of changes in epitopes associated with specific class I alleles was significantly greater than the number of changes in other sites and greater than what was found in that same site for persons who did not possess the allele (Table 3). For HLA B*35 and B*37, sequence changes were 7.0 and 8.5 times as likely to occur in an epitope associated with an allele in women having the allele as compared with those that lacked it (P<0.001). An example of such an epitope is shown in a 38 amino acid region that spans an HLA A2 motif (FIG. 7). Mutations from R to K were noted outside the A2 epitope, and mutations from G to S were noted within the epitope in 8 and 6 of 22 women, respectively. However, while R to K mutation was noted in a similar percentage of A2 positive and A2 negative women (41.7% versus 30:0%, P>0.10), all G to S mutations were observed in A2 positive women (P=0.015), consistent with immune escape as has been observed in the SIV macaque model (10) and chimpanzees infected with HCV (6).

As seen with envelope sequences, the opposite effect was observed in other alleles. For alleles A*01 and B*08, sequence changes were 0.2 and 0.4 times as likely to occur in an epitope restricted by an allele in women having the allele as compared with those who lack it (Table 3). In fact, the R to K mutation that was described above in both A2 positive and negative women, only occurred in women who were not HLA B*08 positive, while the apparently A2-restricted G1409S substitution occurred in both B*08 positive and negative women (FIG. 7).

Collectively, these findings indicate that HCV sequence change is a non-random process that reflects negative selection (change is disadvantageous) as well as positive selection. Moreover, we find evidence that positive selection represents both the direct effect of pressure applied by immune responses in the current host (in this case, HLA class I restricted CD8+ cytotoxic T lymphocytes) as well as reversion of sequence to a more fit consensus, as we saw with envelope sequences.

To independently evaluate this paradigm, we compared the amino acid sequences of these women with a HCV 1b consensus sequence. For the epitopes that showed evidence of HLA class I restricted positive selection (a significantly increased risk of mutations from the inoculum occurred when the restricting allele was present), there was also an increased number of changes away from the 1b prototype consensus in women with one of these alleles, but not those without (Table 4). In addition, for epitopes that showed the converse effect, i.e., evidence of positive selection when the allele was absent (a significantly lower risk of mutations from the inoculum when the restricting allele was present), there was also an increased number of changes toward consensus in those who lacked the allele versus those who had the allele, suggesting reversion (Table 4). These findings are supported in an accompanying report (Cox, et al. J Exp Med. 2005 Jun. 6; 201(11):1741-52 and Example 2), which shows that amino acid substitutions in CD8+ T cell epitopes are associated with a loss of T cell recognition during acute infection, whereas non-epitope changes revert toward consensus at a rate much higher than expected by chance.

Prior studies have demonstrated reversion of CTL-escape variant sequences in macaques experimentally infected with SIV (11), reversion of an HIV-1 epitope in humans (12), and evidence of HIV-1 adaptation to common HLA alleles (13). This is the first report of viral adaptation to multiple HLA alleles across multiple genes, and provides additional support for the suggestion, based on minimizing differences between vaccine and circulating strains, that vaccine effectiveness may be enhanced by using a consensus (14) or ancestral (15) sequence. The ability of viruses to restrict adaptive immune responses and evade those that are formed contributes to persistence and is a major barrier to vaccine development. These data demonstrate that although immune responses diminish the fitness of viral variants, viral divergence occurs in persistently infected hosts. Nonetheless, this divergence actually reduces the fitness of the virus in the population (that is, in other hosts). From an evolutionary perspective, these forces maintain the virus as a distinct pathogen. However, the data also suggest that immune responses to consensus sequences (rather than a product based on the sequence in a given host) would establish the highest barrier to viral escape and consequently the most effective protection against chronic infection.

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TABLE 1 Non-epitope, non-envelope changes^(a) frequently result in modal residue Change Change relative to Site^(b) Region Subject t₀ → t₆ ^(c) Subtype 1a^(d) modal residue^(e) 6 Core 11 N → K K₁₀₂Y₂ Toward 88 Core 17 S → N N₁₂₈ Toward 840 NS2 21 S → G S₁₀A₁ Away 856 NS2 17 L → Q Q₆H₂L₁ Toward 859 NS2 21 V → E V₉ Away 861 NS2 21 V → I V₇I₁F₁ Away 899 NS2 17 F → L L₉ Toward 908 NS2 13 K → R K₉ Away 945 NS2 12 T → A T₉ Away 1021 NS2 12 K → E K₉ Away 1113 NS3 17 T → A A₇₃T₁P₁V₁ Toward 1278 NS3 18 I → L I₇₈S₁ Away 1314 NS3 12 V → I I₄₉F₁ Toward 1338 NS3 18 S → T T₄₆ Toward 1600 NS3 21 P→ L P₅₀L₁ Away 1648 NS3 28 G → C C₅₄Y₁ Toward ^(a)Changes that were not located in demonstrated epitopes and not located in the envelope region (FIG. 2) ^(b)Position in H77 polyprotein (Genbank accession AF009606) ^(c)amino acids inferred from cDNA clones obtained at t₀ and t₆, respectively. Changes were only inferred when independent cDNA clone sequences were in agreement.

TABLE 2 Observed versus expected frequencies of change to modal residue at 16 sites not located in epitopes nor in envelope (E1 or E2) genes Expected Observed Change to modal residue 16 * (1/19) = 0.8 ≈ 1 8* Change to non-modal residue 16 * (18/19) = 15.2 ≈ 15 8  *p = 0.015

TABLE 3 Proportion of amino acid substitutions in known epitopes located in HCV Core, p7, NS2, and NS3, according to the presence or absence of the associated HLA allele (24). Epitopes were considered in the region sequenced (corresponding to amino acid residues 1-1651 of the H77 polyprotein, GenBank AF009606). Two sequences per subject were examined. P values were calculated by comparison of proportions as implemented in SigmaStat (Systat Software, Inc., Richmond, Calif. USA). NA means not applicable due to zero cell. EpiChanges, a software program created by S.C.R. to automate this analysis, is available from the author. Relative Risk of Subjects Allele present Allele absent change (allele with allele Epitopes Epitope changes/ Epitope present versus Allele (n) (n) sites (%) changes/sites absent) P value A*01 11 1 2/198 (1.0) 10/198 (5.1) 0.2 0.019 A*02 13 13 39/3198 (1.2) 18/2214 (0.8) 1.5 >0.10 A*03 9 4 8/648 (1.2) 18/936 (1.9) 0.6 >0.10 A*11 3 3 6/162 (3.7) 45/1026 (4.4) 0.8 >0.10 A*24 1 2 0/36 (0.0) 3/756 (0.4) 0 >0.10 B*07 7 3 4/378 (1.1) 0/810 (0.0) NA 0.003 B*08 6 3 4/312 (1.3) 28/832 (3.4) 0.4 0.057 B*35 3 3 11/162 (6.8) 10/1026 (1.0) 7.0 <0.001 B*37 5 1 20/80 (25.0) 8/272 (2.9) 8.5 <0.001 B*44 7 1 0/126 (0.0) 2/270 (0.7) 0 >0.10

TABLE 4 Classification of amino acid substitutions in MHC class I restricted epitopes as being toward or away from a 1b consensus sequence in 22 women exposed to a common HCV subtype 1b inoculum. Results are stratified (in columns) by the presence or the absence of the relevant allele, and grouped (rows) according to relative risk for change shown in Table 1. Changes are per 1000 epitope sites, based on whether the resulting residue matches HCV 1b consensus. Changes in epitopes relative to Effect of 1b consensus, per 1000 sites allele presence on Allele present Allele absent likelihood of amino Toward Away from Toward Away from acid replacement consensus consensus consensus consensus 620 sites 2108 sites Increased relative 1.6 54.8* 3.9 0.0 risk (B*07, B*35, B*37) 510 sites 1030 sites Decreased relative 3.8 4.7 22.3* 48 risk (A*01, B*08) *P < 0.001 for comparison of Toward versus Away.

TABLE 5 HCV 1a Consensus Sequences A B C D E F G H I J K L M N O P Q R S T U V W 1 M₄₀ 1505 G₄₉ 2 S₄₀ 1506 E₄₉ 3 T₃₉ R₁ 1507 R₄₉ 4 N₃₆ I₄ 1508 P₄₉ 5 P₃₉ L₁ 1509 S₄₇ X₂ 6 K₄₀ 1510 G₄₉ 7 P₃₉ L₁ 1511 M₄₈ X₁ 8 Q₄₀ 1512 F₄₈ X₁ 9 R₄₀ 1513 D₄₉ 10 K₃₇ Q₂ R₁ 1514 S₄₉ 11 T₄₀ 1515 S₄₈ A₁ 12 K₄₆ 1516 V₄₉ 13 R₄₆ 1517 L₄₉ 14 N₄₆ 1518 C₄₉ 15 T₄₆ 1519 E₄₈ X₁ 16 N₄₅ S₁ 1520 C₄₉ 17 R₄₆ 1521 Y₄₉ 18 R₄₆ 1522 D₄₉ 19 P₄₆ 1523 A₄₈ T₁ 20 Q₄₆ 1524 G₄₉ 21 D₄₆ 1525 C₄₈ X₁ 22 V₄₆ 1526 A₄₈ X₁ 23 K₄₆ 1527 W₄₉ 24 F₄₆ 1528 Y₄₈ X₁ 25 P₄₆ 1529 E₄₉ 26 G₄₆ 1530 L₄₉ 27 G₄₆ 1531 T₄₇ X₁ M₁ 28 G₄₆ 1532 P₄₉ 29 Q₄₆ 1533 A₄₇ T₁ X₁ 30 I₄₆ 1534 E₄₉ 31 V₄₆ 1535 T₄₈ X₁ 32 G₄₆ 1536 T₄₉ 33 G₄₆ 1537 V₄₈ A₁ 34 V₄₅ I₁ 1538 R₄₉ 35 Y₄₆ 1539 L₄₉ 36 L₄₆ 1540 R₄₉ 37 L₄₆ 1541 A₄₈ X₁ 38 P₄₆ 1542 Y₄₉ 39 R₄₆ 1543 M₄₉ 40 R₄₅ W₁ 1544 N₄₉ 41 G₄₆ 1545 T₄₉ 42 P₄₆ 1546 P₄₉ 43 R₄₅ K₁ 1547 G₄₉ 44 L₄₆ 1548 L₄₉ 45 G₄₆ 1549 P₄₉ 46 V₄₆ 1550 V₄₉ 47 R₄₆ 1551 C₄₈ X₁ 48 A₄₅ T₁ 1552 Q₄₈ 49 T₄₆ 1553 D₄₈ 50 R₄₆ 1554 H₄₈ 51 K₄₆ 1555 L₄₈ 52 T₄₆ 1556 E₄₈ 53 S₄₆ 1557 F₄₈ 54 E₄₆ 1558 W₄₈ 55 R₄₆ 1559 E₄₈ 56 S₄₆ 1560 G₄₈ 57 Q₄₆ 1561 V₄₈ 58 P₄₆ 1562 F₄₈ 59 R₄₆ 1563 T₄₈ 60 G₄₆ 1564 G₄₇ X₁ 61 R₄₅ S₁ 1565 L₄₈ 62 R₄₆ 1566 T₄₈ 63 Q₄₆ 1567 H₄₇ Q₁ 64 P₄₅ L₁ 1568 I₄₈ 65 I₄₅ F₁ 1569 D₄₈ 66 P₄₅ T₁ 1570 A₄₇ X₁ 67 K₄₅ Q₁ 1571 H₄₇ X₁ 68 A₄₃ V₃ 1572 F₄₈ 69 R₄₆ 1573 L₄₇ I₁ 70 R₄₆ 1574 S₄₅ X₃ 71 P₄₃ S₂ A₁ 1575 Q₄₈ 72 E₄₆ 1576 T₄₈ 73 G₄₆ 1577 K₄₈ 74 R₄₆ 1578 Q₄₈ 75 T₄₆ 1579 S₄₅ X₂ G₁ 76 W₄₆ 1580 G₄₇ X₁ 77 A₄₆ 1581 E₄₆ X₂ 78 Q₄₆ 1582 N₄₈ 79 P₄₆ 1583 F₂₄ L₂₃ P₁ 80 G₄₆ 1584 P₄₈ 81 Y₄₆ 1585 Y₄₈ 82 P₄₆ 1586 L₄₈ 83 W₄₆ 1587 V₄₈ 84 P₄₆ 1588 A₄₈ 85 L₄₆ 1589 Y₄₈ 86 Y₄₆ 1590 Q₄₈ 87 G₄₆ 1591 A₄₈ 88 N₄₆ 1592 T₄₇ X₁ 89 E₄₆ 1593 V₄₈ 90 G₄₆ 1594 C₄₈ 91 C₄₆ 1595 A₄₈ 92 G₄₆ 1596 R₄₇ X₁ 93 W₄₆ 1597 A₄₇ S₁ 94 A₄₅ M₁ 1598 Q₄₇ R₁ 95 G₄₆ 1599 A₄₈ 96 W₄₆ 1600 P₄₆ L₂ 97 L₄₆ 1601 P₄₈ 98 L₄₆ 1602 P₄₇ X₁ 99 S₄₆ 1603 S₄₆ X₂ 100 P₄₆ 1604 W₄₈ 101 R₄₆ 1605 D₄₈ 102 G₄₆ 1606 Q₄₈ 103 S₄₆ 1607 M₄₈ 104 R₄₆ 1608 W₄₈ 105 P₄₆ 1609 K₄₈ 106 S₄₄ N₂ 1610 C₄₈ 107 W₄₆ 1611 L₄₈ 108 G₄₆ 1612 I₃₆ T₈ V₂ X₂ 109 P₄₅ X₁ 1613 R₄₈ 110 T₄₆ 1614 L₄₇ X₁ 111 D₄₆ 1615 K₄₇ X₁ 112 P₄₅ S₁ 1616 P₄₇ X₁ 113 R₄₄ P₂ 1617 T₄₇ 114 R₄₆ 1618 L₄₆ X₂ 115 R₄₄ K₂ 1619 H₄₇ Y₁ 116 S₄₆ 1620 G₄₈ 117 R₄₉ 1621 P₄₆ S₁ X₁ 118 N₄₉ 1622 T₄₇ K₁ 119 L₄₉ 1623 P₄₇ X₁ 120 G₄₉ 1624 L₄₇ X₁ 121 K₄₉ 1625 L₄₇ X₁ 122 V₄₉ 1626 Y₄₈ 123 I₄₉ 1627 R₄₈ 124 D₄₉ 1628 L₄₇ X₁ 125 T₄₉ 1629 G₄₆ X₂ 126 L₄₈ F₁ 1630 A₄₈ 127 T₄₉ 1631 V₄₈ 128 C₄₉ 1632 Q₄₈ 129 G₅₁ 1633 N₄₇ H₁ 130 F₅₀ L₁ 1634 E₄₈ 131 A₅₁ 1635 V₄₀ I₇ X₁ 132 D₅₁ 1636 T₄₇ X₁ 133 L₅₀ H₁ 1637 L₄₅ M₁ 134 M₅₀ T₁ 1638 T₄₆ 135 G₅₁ 1639 H₄₆ 136 Y₅₁ 1640 P₄₅ X₁ 137 I₅₁ 1641 V₂₄ I₂₁ X₁ 138 P₅₁ 1642 T₄₆ 139 L₅₁ 1643 K₄₅ X₁ 140 V₅₁ 1644 Y₄₄ X₁ 141 G₅₁ 1645 I₄₅ 142 A₄₈ V₂ P₁ 1646 M₄₅ 143 P₅₁ 1647 T₄₅ 144 L₅₁ 1648 C₄₅ 145 G₅₀ X₁ 1649 M₄₄ X₁ 146 G₅₁ 1650 S₄₃ A₁ 147 A₅₀ R₁ 1651 A₄₄ 148 A₅₁ 1652 D₄₄ 149 R₄₉ K₂ 1653 L₄₄ 150 A₅₁ 1654 E₄₂ X₂ 151 L₅₁ 1655 V₄₁ I₃ 152 A₅₁ 1656 V₄₃ T₁ 153 H₅₁ 1657 T₄₃ X₁ 154 G₅₀ X₁ 1658 S₄₄ 155 V₅₁ 1659 T₄₄ 156 R₅₁ 1660 W₄₄ 157 V₅₁ 1661 V₄₄ 158 L₅₁ 1662 L₄₃ X₁ 159 E₅₁ 1663 V₄₄ 160 D₅₁ 1664 G₄₄ 161 G₅₀ S₁ 1665 G₄₄ 162 V₅₁ 1666 V₄₄ 163 N₅₁ 1667 L₄₄ 164 Y₅₁ 1668 A₄₄ 165 A₅₁ 1669 A₄₄ 166 T₅₁ 1670 L₄₄ 167 G₅₁ 1671 A₄₃ X₁ 168 N₅₁ 1672 A₄₄ 169 L₅₁ 1673 Y₄₄ 170 P₅₁ 1674 C₄₄ 171 G₅₁ 1675 L₄₄ 172 C₅₂ 1676 S₄₄ 173 S₅₂ 1677 T₄₃ X₁ 174 F₅₁ X₁ 1678 G₄₄ 175 S₅₁ 1679 C₄₃ S₁ 176 I₄₉ X₁₂ M₁ L₁ 1680 V₄₄ 177 F₇₂ 1681 V₄₄ 178 L₇₂ X₁ 1682 I₄₄ 179 L₇₃ X₁ 1683 V₄₃ I₁ 180 A₇₄ X₅ 1684 G₄₄ 181 L₇₉ 1685 R₄₄ 182 L₇₈ F₁ 1686 I₂₅ V₁₈ X₁ 183 S₈₀ 1687 V₄₀ I₂ D₁ X₁ 184 C₈₀ 1688 L₄₄ 185 L₈₀ 1689 S₄₄ 186 T₇₈ A₁ I₁ 1690 G₄₄ 187 V₇₉ I₁ X₁ 1691 K₄₄ 188 P₈₁ 1692 P₄₄ 189 A₈₀ T₁ 1693 A₄₃ P₁ 190 S₈₀ W₁ 1694 I₃₄ V₇ X₃ 191 A₈₀ P₁ 1695 I₄₃ V₁ 192 Y₈₈ H₆ F₁ 1696 P₄₄ 193 Q₈₉ H₃ E₃ 1697 D₄₄ 194 V₉₅ 1698 R₄₂ Q₁ X₁ 195 R₉₅ 1699 E₄₃ 196 N₉₃ 1700 V₄₃ A₁ 197 S₉₁ T₁ A₁ 1701 L₄₄ 198 S₄₈ T₄₄ L₁ 1702 Y₄₃ X₁ 199 G₉₃ 1703 R₃₀ Q₁₄ 200 L₈₇ I₄ F₁ S₁ 1704 E₄₂ G₂ 201 Y₉₃ 1705 F₄₄ 202 H₉₃ 1706 D₄₄ 203 V₉₃ X₁ 1707 E₄₄ 204 T₉₄ 1708 M₄₄ 205 N₉₃ X₁ 1709 E₄₄ 206 D₉₂ H₂ 1710 E₄₄ 207 C₉₄ 1711 C₄₄ 208 P₉₂ S₁ L₁ 1712 S₄₄ 209 N₉₄ 1713 Q₄₄ 210 S₉₄ 1714 H₄₄ 211 S₉₃ G₁ 1715 L₄₄ 212 I₉₃ V₁ 1716 P₄₃ X₁ 213 V₉₄ 1717 Y₄₄ 214 Y₉₂ F₂ 1718 I₄₄ 215 E₉₄ 1719 E₄₄ 216 A₅₂ T₄₁ V₁ 1720 Q₄₃ P₁ 217 A₆₇ D₁₄ T₅ V₂ S₂ H₁ P₁ N₁ G₁ 1721 G₄₄ 218 D₉₀ N₃ G₁ 1722 M₄₄ 219 A₆₅ T₂₇ V₁ S₁ 1723 M₄₀ A₂ V₁ X₁ 220 I₉₄ 1724 L₄₃ X₁ 221 L₉₃ M₁ 1725 A₄₃ X₁ 222 H₉₄ 1726 E₄₃ X₁ 223 T₅₂ S₃₁ A₉ I₁ V₁ 1727 Q₄₄ 224 P₉₄ 1728 F₄₄ 225 G₉₄ 1729 K₄₄ 226 C₉₄ 1730 Q₄₄ 227 V₈₉ I₃ F₁ L₁ 1731 K₄₃ X₁ 228 P₉₄ 1732 A₄₄ 229 C₉₄ 1733 L₄₁ X₃ 230 V₉₂ X₁ A₁ 1734 G₄₂ A₁ X₁ 231 R₈₆ H₅ C₁ G₁ Y₁ 1735 L₄₄ 232 E₉₁ K₂ G₁ 1736 L₄₄ 233 G₉₀ D₃ S₁ 1737 Q₄₄ 234 N₉₀ D₃ S₁ 1738 T₄₃ X₁ 235 A₅₅ T₁₅ V₁₃ I₄ S₂ D₂ L₁ X₁ G₁ 1739 A₄₃ R₁ 236 S₉₁ P₂ A₁ 1740 S₄₄ 237 R₆₄ K₂₉ T₁ 1741 R₃₉ H₃ X₂ 238 C₉₄ 1742 Q₄₂ H₂ 239 W₉₄ 1743 A₄₃ X₁ 240 V₉₃ A₁ 1744 E₄₄ 241 A₈₇ P₅ T₁ S₁ 1745 V₃₇ A₃ X₃ T₁ 242 V₅₄ M₂₈ L₈ I₄ 1746 I₄₁ V₁ X₁ 243 T₆₂ A₃₂ 1747 A₂₅ T₁₅ G₂ X₁ 244 P₉₃ S₁ 1748 P₄₂ X₁ 245 T₉₂ S₂ 1749 A₃₈ V₃ T₂ 246 V₉₃ L₁ 1750 V₄₃ 247 A₉₄ 1751 Q₄₃ 248 T₉₃ A₁ 1752 T₄₃ 249 R₈₆ K₈ 1753 N₄₁ S₂ 250 D₉₂ N₂ 1754 W₄₂ R₁ 251 G₉₃ A₁ 1755 Q₄₂ X₁ 252 K₇₅ R₁₃ S₂ N₂ T₁ X₁ 1756 K₃₄ R₇ N₁ X₁ 253 L₉₂ I₁ V₁ 1757 L₄₂ I₁ 254 P₉₃ H₁ 1758 E₃₉ V₁ X₁ 255 T₇₇ A₁₅ S₁ I₁ 1759 A₂₁ T₁₁ V₆ X₂ F₁ S₁ L₁ 256 T₈₆ A₅ K₂ M₁ 1760 F₃₇ G₂ V₁ L₁ I₁ X₁ 257 Q₉₃ T₁ 1761 W₃₇ G₆ X₁ 258 L₉₂ I₁ M₁ 1762 A₄₁ G₂ K₁ 259 R₉₄ 1763 K₄₃ X₂ M₁ 260 R₉₃ Q₁ 1764 H₄₂ N₂ K₂ 261 H₉₂ X₁ Y₁ 1765 X₂₇ M₁₉ 262 I₉₃ V₁ 1766 W₁₈ X₁ 263 D₉₄ 1767 N₁₈ 264 L₉₃ X₁ 1768 F₁₇ X₁ 265 L₉₃ X₁ 1769 I₁₆ X₁ 266 V₉₃ G₁ 1770 S₁₇ 267 G₉₃ R₁ 1771 G₁₇ 268 S₇₅ G₁₆ A₂ X₁ 1772 I₁₇ 269 A₉₄ 1773 Q₁₇ 270 T₉₂ A₂ 1774 Y₁₇ 271 L₉₁ F₂ I₁ 1775 L₁₇ 272 C₉₄ 1776 A₁₇ 273 S₉₄ 1777 G₁₇ 274 A₉₂ T₂ 1778 L₁₇ 275 L₉₃ M₁ 1779 S₁₇ 276 Y₉₇ 1780 T₁₇ 277 V₉₇ 1781 L₁₇ 278 G₉₇ 1782 P₁₆ A₁ 279 D₉₆ E₁ 1783 G₁₇ 280 L₉₇ 1784 N₁₇ 281 C₉₇ 1785 P₁₇ 282 G₉₇ 1786 A₁₇ 283 S₉₇ 1787 I₁₄ X₃ 284 V₉₂ I₅ 1788 A₁₄ 285 F₉₅ L₂ 1789 S₁₄ 286 L₉₇ 1790 L₁₄ 287 V₉₃ I₃ L₁ 1791 M₁₄ 288 G₉₁ S₅ D₁ 1792 A₁₄ 289 Q₉₇ 1793 F₁₄ 290 L₉₅ M₂ 1794 T₁₄ 291 F₉₇ 1795 A₁₄ 292 T₉₆ V₁ 1796 A₁₄ 293 F₉₂ L₄ I₁ 1797 V₁₃ I₁ 294 S₉₇ 1798 T₁₄ 295 P₉₇ 1799 S₁₄ 296 R₉₅ G₁ K₁ 1800 P₁₄ 297 R₈₇ H₈ L₁ D₁ 1801 L₁₄ 298 H₉₂ Y₃ R₁ L₁ 1802 T₁₄ 299 W₉₅ R₁ E₁ 1803 T₁₄ 300 T₉₅ Q₁ A₁ 1804 S₁₀ G₃ N₁ 301 T₉₄ V₂ M₁ 1805 Q₁₄ 302 Q₉₇ 1806 T₁₄ 303 D₆₅ G₂₃ E₇ S₁ R₁ 1807 L₁₄ 304 C₉₇ 1808 L₁₄ 305 N₉₇ 1809 F₁₄ 306 C₉₇ 1810 N₁₄ 307 S₉₆ T₁ 1811 I₁₄ 308 I₈₃ M₁₀ L₂ V₂ 1812 L₁₄ 309 Y₉₆ 1813 G₁₄ 310 P₉₆ 1814 G₁₃ S₁ 311 G₉₅ S₁ 1815 W₁₄ 312 H₉₅ D₁ 1816 V₁₄ 313 I₉₁ V₄ L₁ 1817 A₁₄ 314 T₉₄ G₁ S₁ 1818 A₁₄ 315 G₉₆ 1819 Q₁₄ 316 H₉₆ 1820 L₁₄ 317 R₉₅ G₁ 1821 A₁₄ 318 M₁₀₉ 1822 A₁₂ N₁ G₁ 319 A₁₀₉ 1823 P₁₄ 320 W₁₀₈ 1824 G₁₃ R₁ 321 D₁₀₈ X₁₉ N₁ 1825 A₁₄ 322 M₁₂₈ 1826 A₁₄ 323 M₁₃₃ 1827 T₁₄ 324 M₁₃₀ V₂ T₁ 1828 A₁₄ 325 N₁₃₂ I₁ 1829 F₁₄ 326 W₁₃₄ 1830 V₁₄ 327 S₁₃₄ 1831 G₁₄ 328 P₁₃₄ L₂ X₁ 1832 A₁₄ 329 T₁₃₆ K₁ 1833 G₁₄ 330 T₉₁ A₄₂ I₂ X₁ V₁ 1834 L₁₄ 331 A₁₃₈ 1835 A₁₃ T₁ 332 L₁₃₆ F₁ P₁ 1836 G₁₄ 333 V₁₂₃ I₁₀ L₅ 1837 A₁₄ 334 V₁₀₀ M₂₉ T₃ L₃ A₂ I₁ 1838 A₁₃ G₁ 335 A₁₃₂ S₄ V₁ G₁ 1839 I₁₃ V₁ 336 Q₁₃₈ 1840 G₁₄ 337 L₁₃₂ V₄ M₂ 1841 S₁₃ R₁ 338 L₁₃₅ I₁ F₁ M₁ 1842 V₁₃ D₁ 339 R₁₃₈ 1843 G₁₄ 340 I₁₀₀ V₃₈ 1844 L₁₄ 341 P₁₃₈ 1845 G₁₄ 342 Q₁₃₇ L₁ 1846 K₁₄ 343 A₁₃₆ T₁ S₁ 1847 V₁₄ 344 I₁₃₄ V₃ T₁ 1848 L₁₄ 345 L₁₀₄ M₂₆ V₈ 1849 V₁₃ I₁ 346 D₁₃₆ N₂ 1850 D₁₄ 347 M₁₃₈ I₁ 1851 I₁₄ 348 I₁₃₄ V₃ F₂ 1852 L₁₄ 349 A₁₃₉ 1853 A₁₃ V₁ 350 G₁₃₈ S₁ 1854 G₁₄ 351 A₁₃₈ S₁ 1855 Y₁₄ 352 H₁₃₉ 1856 G₁₄ 353 W₁₃₉ 1857 A₁₄ 354 G₁₃₉ 1858 G₁₄ 355 V₁₃₆ I₃ 1859 V₁₄ 356 L₁₃₉ 1860 A₁₄ 357 A₁₃₉ 1861 G₁₄ 358 G₁₃₉ 1862 A₁₄ 359 I₁₂₃ V₇ M₆ L₃ 1863 L₁₄ 360 A₁₃₄ T₄ G₁ 1864 V₁₄ 361 Y₁₃₉ 1865 A₁₄ 362 F₁₃₆ Y₃ 1866 F₁₄ 363 S₁₃₉ 1867 K₁₄ 364 M₁₃₉ 1868 I₁₄ 365 V₁₃₃ A₅ L₁ 1869 M₁₄ 366 G₁₃₉ 1870 S₁₄ 367 N₁₄₀ 1871 G₁₄ 368 W₁₄₀ 1872 E₁₄ 369 A₁₃₉ G₁ 1873 V₁₃ P₁ 370 K₁₄₀ 1874 P₁₄ 371 V₁₃₉ A₁ 1875 S₁₃ T₁ 372 L₁₂₉ V₁₀ S₁ 1876 T₁₄ 373 V₁₂₇ L₇ I₃ A₃ X₁ 1877 E₁₄ 374 V₁₄₁ X₂ M₁ 1878 D₁₄ 375 L₁₄₄ M₁ 1879 L₁₃ M₁ 376 L₁₄₅ M₁ 1880 V₁₄ 377 L₁₄₆ 1881 N₁₄ 378 F₁₄₅ V₁ 1882 L₁₄ 379 A₁₄₂ T₄ 1883 L₁₃ X₁ 380 G₁₃₅ S₉ A₁ X₁ 1884 P₁₄ 381 V₁₄₅ I₁ 1885 A₁₄ 382 D₁₄₆ Q₁ X₁ 1886 I₁₄ 383 A₁₄₅ G₂ R₁ 1887 L₁₄ 384 E₆₈ G₂₀ T₁₁ S₁₀ D₆ K₅ N₅ Q₄ A₃ H₃ V₂ R₂ W₁ I₁ 1888 S₁₄ 385 T₁₃₉ P₁ X₁ 1889 P₁₄ 386 H₆₅ Y₄₈ R₉ T₈ V₂ I₂ S₁ M₁ D₁ Q₁ L₁ G₁ X₁ 1890 G₁₄ 387 V₉₇ T₃₆ I₄ L₂ A₁ R₁ 1891 A₁₄ 388 T₁₁₂ S₂₄ I₄ V₁ 1892 L₁₄ 389 G₁₄₁ 1893 V₁₄ 390 G₁₃₃ A₆ R₁ D₁ 1894 V₁₄ 391 S₇₅ A₂₃ T₁₉ N₉ V₆ Q₅ G₁ K₁ H₁ E₁ 1895 G₁₄ 392 A₁₀₉ V₂₀ I₄ T₃ P₂ L₁ S₁ X₁ 1896 V₁₄ 393 A₈₈ G₄₉ S₃ V₁ 1897 V₁₄ 394 R₇₅ H₃₇ Q₈ K₆ Y₆ S₃ F₂ E₂ V₁ N₁ 1898 C₁₃ Y₁ 395 T₆₃ A₄₃ S₁₂ G₁₂ D₅ V₂ N₂ I₂ 1899 A₁₄ 396 T₆₉ A₄₄ V₂₀ M₄ S₂ X₁ I₁ 1900 A₁₃ T₁ 397 S₅₀ A₄₈ L₁₀ Y₇ H₆ Q₅ F₄ R₃ V₂ T₂ N₂ K₁ G₁ 1901 I₁₄ 398 G₉₇ S₁₁ T₉ R₉ A₇ V₃ I₃ L₁ M₁ 1902 L₁₃ Q₁ 399 L₇₂ F₄₇ I₁₅ V₇ 1903 R₁₄ 400 A₇₀ T₂₈ V₂₆ S₁₃ I₂ X₁ N₁ 1904 R₁₄ 401 G₅₆ S₅₄ R₁₅ N₆ T₅ D₃ A₁ X₁ 1905 H₁₄ 402 L₉₁ F₃₄ I₁₀ M₃ X₂ P₁ 1906 V₁₄ 403 F₁₁₀ L₃₀ S₁ 1907 G₁₄ 404 S₅₄ T₄₂ A₁₈ N₁₃ Q₄ D₃ K₃ P₁ E₁ R₁ H₁ 1908 P₁₄ 405 P₈₄ Q₂₂ R₁₁ L₇ S₇ T₄ A₃ I₂ V₁ 1909 G₁₄ 406 G₁₄₁ 1910 E₁₄ 407 A₉₄ P₄₁ S₆ 1911 G₁₄ 408 K₈₇ R₂₇ S₁₄ Q₁₁ N₂ 1912 A₁₄ 409 Q₁₄₁ 1913 V₁₄ 410 N₁₀₆ D₂₃ K₉ H₂ X₁ 1914 Q₁₄ 411 I₁₀₇ V₃₂ L₂ 1915 W₁₄ 412 Q₁₂₉ R₈ K₁ H₁ 1916 M₁₄ 413 L₁₄₀ 1917 N₁₄ 414 I₁₀₇ V₂₉ T₂ M₂ 1918 R₁₄ 415 N₁₃₆ K₃ Y₁ 1919 L₁₃ M₁ 416 T₁₁₃ S₂₃ A₄ 1920 I₁₄ 417 N₁₃₆ D₂ E₁ S₁ 1921 A₁₄ 418 G₁₄₀ 1922 F₁₄ 419 S₁₄₀ 1923 A₁₃ T₁ 420 W₁₄₀ 1924 S₁₄ 421 H₁₄₀ 1925 R₁₄ 422 I₁₃₉ V₁ 1926 G₁₄ 423 N₁₃₈ D₁ X₁ 1927 N₁₃ G₁ 424 R₈₁ S₅₉ 1928 H₁₃ X₁ 425 T₁₃₈ A₁ S₁ 1929 V₁₄ 426 A₁₄₀ 1930 S₁₄ 427 L₁₄₀ 1931 P₁₄ 428 N₁₄₀ 1932 T₁₄ 429 C₁₄₀ 1933 H₁₄ 430 N₁₃₇ S₂ D₁ 1934 Y₁₄ 431 D₇₀ A₄₈ E₂₀ T₁ S₁ 1935 V₁₄ 432 S₁₃₇ N₂ T₁ 1936 P₁₄ 433 L₁₃₆ H₂ I₂ 1937 E₁₄ 434 N₅₉ D₄₁ E₁₂ T₉ H₉ Q₄ S₃ Y₁ K₁ X₁ 1938 S₁₃ N₁ 435 T₁₃₅ A₄ S₁ 1939 D₁₄ 436 G₁₄₀ 1940 A₁₃ T₁ 437 W₁₂₈ F₁₂ 1941 A₁₄ 438 I₄₉ L₄₄ V₃₉ M₄ 1942 A₁₃ 439 A₁₂₉ T₄ V₂ G₁ 1943 R₁₃ 440 G₁₂₉ S₃ R₂ A₂ 1944 V₁₃ 441 L₁₃₅ P₁ 1945 T₁₃ 442 F₉₅ L₂₆ I₁₁ S₁ M₁ X₁ V₁ 1946 A₁₀ T₂ V₁ 443 Y₁₃₂ H₄ 1947 I₁₃ 444 Y₇₀ H₄₀ R₈ S₄ Q₄ N₃ V₂ F₂ P₁ T₁ A₁ 1948 L₁₃ 445 H₈₂ N₃₇ Y₉ D₂ R₂ S₁ K₁ T₁ L₁ 1949 S₁₁ G₂ 446 K₁₀₇ R₁₃ Q₅ N₃ H₃ S₂ G₂ T₁ 1950 S₁₃ 447 F₁₃₆ 1951 L₁₃ 448 N₁₃₂ D₃ K₁ 1952 T₁₂ 449 S₁₃₀ F₂ D₂ A₂ 1953 V₁₂ 450 S₁₂₅ T₁₁ X₁ 1954 T₁₁ N₁ 451 G₁₃₇ 1955 Q₉ X₂ K₁ 452 C₁₃₇ 1956 L₉ P₂ 453 P₁₁₁ S₁₅ T₃ A₃ L₂ F₁ R₁ V₁ 1957 L₁₁ 454 E₁₃₄ Q₁ G₁ A₁ 1958 R₉ X₂ 455 R₁₃₅ M₁ G₁ 1959 R₈ X₁ G₁ 456 L₈₁ M₅₄ S₁ V₁ 1960 L₈ 457 A₁₃₄ S₂ T₁ 1961 H₈ 458 S₁₃₅ G₁ T₁ 1962 Q₈ 459 C₁₃₃ S₁ X₁ R₁ Y₁ 1963 W₈ 460 R₁₁₈ K₁₂ Q₃ X₁ H₁ L₁ 1964 I₆ V₂ 461 P₉₆ R₂₈ S₆ L₂ X₁ N₁ 1965 S₈ 462 L₁₂₄ I₄ F₃ V₁ 1966 S₈ 463 T₇₉ A₄₉ S₂ I₁ D₁ 1967 E₈ 464 D₁₁₁ Y₅ N₄ H₄ A₃ S₂ R₁ T₁ G₁ 1968 C₇ S₁ 465 F₁₂₈ L₂ Y₂ 1969 T₈ 466 D₆₉ A₆₀ V₁ S₁ G₁ 1970 S₁₀ T₈ 467 Q₁₃₂ 1971 M₁₀ P₈ 468 G₁₃₂ 1972 G₁₀ C₈ M₃ 469 W₁₃₀ S₁ R₁ 1973 S₂₂ A₃ 470 G₁₃₁ X₁ 1974 G₂₅ 471 P₁₃₁ L₁ 1975 S₂₅ 472 I₁₃₁ M₁ 1976 W₂₅ 473 S₉₉ G₁₂ R₇ T₆ N₂ I₂ X₁ D₁ K₁ E₁ 1977 L₂₅ I₁ 474 Y₈₄ H₄₇ F₁ 1978 R₂₆ 475 A₉₉ T₂₁ V₈ G₂ N₁ I₁ 1979 D₂₆ 476 N₁₁₀ D₁₇ T₂ E₂ Y₁ 1980 I₂₆ 477 G₁₂₅ T₂ V₁ R₁ P₁ I₁ Q₁ 1981 W₂₆ 478 S₁₁₁ G₉ T₇ N₄ D₁ 1982 D₂₆ 479 G₁₂₄ D₃ S₂ N₁ R₁ E₁ 1983 W₂₆ 480 P₁₁₀ S₁₁ L₈ F₁ R₁ A₁ 1984 I₂₆ 481 D₈₇ E₄₀ A₂ G₂ K₁ 1985 C₄₇ 482 Q₅₇ H₅₃ E₂₁ G₁ 1986 E₄₇ 483 R₁₃₂ 1987 V₄₇ 484 P₁₃₂ 1988 L₄₆ V₁ 485 Y₁₃₁ H₁ 1989 S₄₆ C₁ 486 C₁₃₂ 1990 D₄₇ 487 W₁₃₁ 1991 F₄₇ 488 H₁₃₂ 1992 K₄₆ E₁ 489 Y₁₃₀ C₁ H₁ 1993 T₄₇ 490 P₁₀₃ T₁₆ S₆ A₆ X₁ 1994 W₄₇ 491 P₁₁₁ X₂₀ T₁ 1995 L₂₆ X₂₁ 492 K₇₇ R₃₅ L₁ 1996 K₄₇ 493 P₁₀₉ R₁ L₁ S₁ 1997 A₄₇ 494 C₁₁₀ W₁ S₁ 1998 K₄₆ Q₁ 495 G₁₁₁ D₁ 1999 L₄₇ 496 I₁₀₃ X₅ V₂ F₁ T₁ 2000 M₄₆ V₁ 497 V₁₀₂ E₂ X₁ G₁ 2001 P₄₇ 498 P₁₀₀ S₃ X₁ D₁ 2002 Q₄₆ R₁ 499 A₁₀₀ X₃ T₁ 2003 L₄₇ 500 K₄₇ Q₂₆ R₁₄ E₃ L₃ M₂ X₂ G₂ S₁ 2004 P₄₇ 501 S₅₁ T₂₀ N₁₉ Q₂ G₂ X₁ R₁ A₁ 2005 G₄₇ 502 V₉₃ D₁ X₁ I₁ 2006 I₄₆ L₁ 503 C₇₂ W₁ X₁ 2007 P₄₇ 504 G₆₇ A₁ 2008 F₄₂ L₅ 505 P₆₅ R₂ Q₁ 2009 V₄₆ M₁ 506 V₆₅ X₃ 2010 S₄₇ 507 Y₅₈ F₁ V₁ 2011 C₄₇ 508 C₅₇ Y₁ G₁ X₁ 2012 Q₄₇ 509 F₅₃ X₇ 2013 R₄₆ Q₁ 510 T₅₂ A₁ 2014 G₄₇ 511 P₅₂ L₁ 2015 Y₄₇ 512 S₅₃ 2016 R₃₃ K₁₄ 513 P₅₃ 2017 G₂₆ X₂₁ 514 V₅₁ 2018 V₄₅ A₂ 515 V₄₈ A₃ 2019 W₄₇ 516 V₅₁ 2020 R₄₆ Q₁ 517 G₅₁ 2021 G₄₄ V₂ A₁ 518 T₅₁ 2022 D₄₆ E₁ 519 T₅₁ 2023 G₄₇ 520 D₄₇ N₄ 2024 I₄₃ V₄ 521 R₃₅ K₁₄ G₁ H₁ 2025 M₄₇ 522 S₃₃ L₁₁ A₄ F₁ M₁ T₁ 2026 H₄₆ Y₁ 523 G₅₁ 2027 T₄₇ 524 A₃₈ V₁₁ M₁ T₁ 2028 R₄₆ H₁ 525 P₅₀ A₁ 2029 C₄₇ 526 T₅₁ 2030 H₄₃ Y₂ N₁ P₁ 527 Y₅₁ 2031 C₄₆ 528 N₃₂ S₁₅ R₃ T₁ 2032 G₄₆ Q₁ 529 W₅₁ 2033 A₄₆ T₁ 530 G₅₁ 2034 E₄₅ A₁ D₁ 531 E₂₄ A₁₅ S₁₀ V₁ Q₁ 2035 I₄₆ G₁ 532 N₅₀ T₁ 2036 T₄₄ A₂ S₁ 533 D₃₉ E₁₁ R₁ 2037 G₄₇ 534 T₅₀ A₁ 2038 H₄₇ 535 D₅₁ 2039 V₄₇ 536 V₄₉ I₁ F₁ 2040 K₂₉ X₁₈ 537 F₄₄ L₇ 2041 N₄₄ T₂ M₁ 538 V₄₅ I₄ L₁ X₁ 2042 G₂₆ X₂₁ 539 L₅₁ 2043 T₄₆ S₁ 540 N₅₀ T₁ 2044 M₄₇ 541 N₄₉ Y₁ S₁ 2045 R₄₇ 542 T₅₀ X₁ 2046 I₄₅ T₁ V₁ 543 R₅₀ 2047 V₄₀ A₄ I₂ F₁ 544 P₅₀ 2048 G₄₇ 545 P₄₉ R₁ 2049 P₄₇ 546 L₄₂ M₃ S₂ Q₁ G₁ W₁ 2050 R₂₄ K₂₃ 547 G₄₉ A₁ 2051 T₄₇ 548 N₄₈ G₁ L₁ 2052 C₄₇ 549 W₄₈ V₁ 2053 R₄₆ K₁ 550 F₄₆ S₂ G₁ 2054 N₄₇ 551 G₄₅ X₃ 2055 M₄₄ T₁ V₁ I₁ 552 C₄₁ X₂ 2056 W₂₆ X₂₁ 553 T₄₀ N₁ 2057 S₃₉ N₇ D₁ 554 W₄₁ 2058 G₄₇ 555 M₄₁ 2059 T₄₆ A₁ 556 N₄₁ 2060 F₄₅ L₂ 557 S₄₀ A₁ 2061 P₄₆ L₁ 558 T₂₇ S₁₃ C₁ 2062 I₄₇ 559 G₄₁ 2063 N₄₇ 560 F₃₃ Y₈ 2064 A₄₆ P₁ 561 T₄₁ 2065 Y₄₇ 562 K₄₁ 2066 T₄₇ 563 V₃₆ T₃ A₂ 2067 T₄₇ 564 C₄₁ 2068 G₄₇ 565 G₄₁ 2069 P₄₇ 566 A₄₀ G₁ 2070 C₄₇ 567 P₄₁ 2071 T₄₄ N₂ V₁ 568 P₄₁ 2072 P₄₇ 569 C₄₁ 2073 L₂₆ X₁₈ S₃ 570 V₂₃ A₆ D₅ N₄ T₂ F₁ 2074 P₄₄ X₃ 571 I₄₁ 2075 A₄₇ 572 G₃₉ R₁ K₁ 2076 P₄₇ 573 G₄₁ 2077 N₄₅ S₂ 574 V₂₄ A₇ G₅ M₂ S₁ K₁ I₁ 2078 Y₄₇ 575 G₃₈ S₂ N₁ 2079 T₃₀ K₁₃ S₃ E₁ 576 N₄₁ 2080 F₄₇ 577 N₃₇ T₂ D₁ H₁ 2081 A₄₇ 578 T₄₀ F₁ 2082 L₄₇ 579 L₄₀ W₁ 2083 W₄₇ 580 H₂₃ Y₇ R₄ L₃ S₂ Q₁ T₁ 2084 R₂₆ X₂₁ 581 C₄₁ 2085 V₄₇ 582 P₄₁ 2086 S₄₇ 583 T₄₁ 2087 A₄₇ 584 D₄₁ 2088 E₄₇ 585 C₄₁ 2089 E₄₆ D₁ 586 F₄₁ 2090 Y₄₇ 587 R₄₁ 2091 V₄₆ A₁ 588 K₄₁ 2092 E₄₆ A₁ 589 H₄₁ 2093 I₄₄ V₃ 590 P₄₁ 2094 R₂₆ X₂₁ 591 E₄₀ D₁ 2095 Q₃₅ R₁₂ 592 A₄₁ 2096 V₂₇ X₂₀ 593 T₄₁ 2097 G₄₆ X₁ 594 Y₄₁ 2098 D₄₇ 595 S₃₉ A₂ 2099 F₄₇ 596 R₄₀ K₁ 2100 H₄₇ 597 C₄₁ 2101 Y₄₇ 598 G₄₁ 2102 V₄₇ 599 S₄₁ 2103 T₄₅ S₁ V₁ 600 G₄₁ 2104 G₄₇ 601 P₄₀ A₁ 2105 M₄₆ V₁ 602 W₄₁ 2106 T₄₇ 603 I₃₄ L₆ V₁ 2107 T₃₉ A₈ 604 T₄₁ 2108 D₄₇ 605 P₄₁ 2109 N₄₃ D₄ 606 R₄₀ K₁ 2110 L₄₇ 607 C₄₁ 2111 K₄₄ R₃ 608 L₃₇ M₃ I₁ 2112 C₄₇ 609 V₄₁ 2113 P₄₇ 610 H₁₆ D₁₄ N₁₁ 2114 C₄₇ 611 Y₄₁ 2115 Q₄₇ 612 P₃₈ A₂ S₁ 2116 V₄₆ I₁ 613 Y₄₁ 2117 P₄₇ 614 R₄₁ 2118 S₄₅ T₁ A₁ 615 L₄₁ 2119 P₄₇ 616 W₃₈ 2120 E₄₇ 617 H₃₈ 2121 F₄₇ 618 X₂₅ Y₁₃ 2122 F₄₇ 619 P₁₃ 2123 T₄₇ 620 C₁₃ 2124 E₄₇ 621 T₁₃ 2125 L₄₆ V₁ 622 I₈ V₂ L₂ M₁ 2126 D₄₇ 623 N₁₃ 2127 G₄₇ 624 Y₁₂ F₁ 2128 V₄₇ 625 T₁₂ S₁ 2129 R₄₇ 626 I₈ L₄ T₁ 2130 L₄₅ I₂ 627 F₁₃ 2131 H₄₇ 628 K₁₃ 2132 R₂₆ X₂₁ 629 V₉ I₄ 2133 F₂₆ X₂₁ 630 R₁₃ 2134 A₄₇ 631 M₁₃ 2135 P₄₇ 632 Y₁₃ 2136 P₄₇ 633 V₁₃ 2137 C₄₇ 634 G₁₃ 2138 K₄₇ 635 G₁₃ 2139 P₄₇ 636 V₁₃ 2140 L₄₇ 637 E₁₃ 2141 L₄₇ 638 H₁₃ 2142 R₄₆ A₁ 639 R₁₃ 2143 E₃₁ D₁₆ 640 L₁₃ 2144 E₄₇ 641 E₈ G₁ Q₁ D₁ N₁ 2145 V₂₈ X₁₉ 642 A₁₁ V₁ 2146 S₂₄ T₁₅ A₆ X₂ 643 A₁₂ 2147 F₂₇ X₂₀ 644 C₁₂ 2148 R₄₆ S₁ 645 N₁₂ 2149 V₄₇ 646 W₁₂ 2150 G₄₇ 647 T₁₂ 2151 L₄₇ 648 R₁₂ 2152 H₄₆ N₁ 649 G₁₂ 2153 E₂₄ D₁₅ A₅ S₁ V₁ T₁ 650 E₁₂ 2154 Y₄₇ 651 R₁₂ 2155 P₄₇ 652 C₁₂ 2156 V₂₈ X₁₉ 653 D₁₀ N₂ 2157 G₄₅ X₂ 654 L₁₂ 2158 S₄₇ 655 E₉ D₃ 2159 Q₄₇ 656 D₁₂ 2160 L₄₇ 657 R₁₂ 2161 P₄₇ 658 D₁₂ 2162 C₄₇ 659 R₁₂ 2163 E₄₇ 660 S₁₂ 2164 P₄₇ 661 E₁₂ 2165 E₄₇ 662 L₁₂ 2166 P₂₆ X₂₁ 663 S₁₂ 2167 D₄₇ 664 P₁₂ 2168 V₄₇ 665 L₁₂ 2169 A₄₅ T₂ 666 L₁₂ 2170 V₄₇ 667 L₁₂ 2171 L₄₅ V₂ 668 S₁₀ T2 2172 T₄₇ 669 T₁₂ 2173 S₄₇ 670 T₁₂ 2174 M₄₆ T₁ 671 Q₁₁ E₁ 2175 L₄₇ 672 W₁₂ 2176 T₄₇ 673 Q₁₂ 2177 D₄₇ 674 V₉ I₃ 2178 P₄₆ S₁ 675 L₁₂ 2179 S₄₇ 676 P₁₂ 2180 H₄₇ 677 C₁₂ 2181 I₄₄ V₂ L₁ 678 S₁₂ 2182 T₄₇ 679 F₁₂ 2183 A₄₇ 680 T₁₂ 2184 E₄₇ 681 T₁₂ 2185 A₂₅ X₂₁ T₁ 682 L₁₂ 2186 A₄₇ 683 P₁₂ 2187 G₃₃ R₇ K₄ A₃ 684 A₁₂ 2188 R₄₇ 685 L₁₂ 2189 R₄₇ 686 S₉ T₃ 2190 L₄₇ 687 T₁₂ 2191 A₄₁ X₄ E₁ T₁ 688 G₁₂ 2192 R₃₀ X₁₇ 689 L₁₂ 2193 G₄₇ 690 I₁₂ 2194 S₄₇ 691 H₁₂ 2195 P₄₆ S₁ 692 L₁₂ 2196 P₃₃ X₁₃ L₁ 693 H₁₂ 2197 S₂₆ X₁₆ 694 Q₁₂ 2198 V₂₄ E₁₁ M₆ L₃ X₃ 695 N₁₂ 2199 A₄₇ 696 I₁₂ 2200 S₄₇ 697 V₁₂ 2201 S₄₇ 698 D₁₂ 2202 S₄₇ 699 V₁₂ 2203 A₄₆ V₁ 700 Q₁₂ 2204 S₄₇ 701 Y₁₂ 2205 Q₄₇ 702 L₁₂ 2206 L₄₇ 703 Y₁₂ 2207 S₄₇ 704 G₁₂ 2208 A₄₆ T₁ 705 V₁₁ I₁ 2209 P₄₆ L₁ 706 G₁₂ 2210 S₄₆ P₁ 707 S₁₂ 2211 L₄₇ 708 S₁₁ A₁ 2212 K₄₆ R₁ 709 I₈ V₄ 2213 A₄₇ 710 A₇ V₄ T₁ 2214 T₄₆ A₁ 711 S₁₂ 2215 C₄₇ 712 W₁₁ Y₁ 2216 T₄₇ 713 A₁₂ 2217 A₂₄ T₁₇ V₃ I₂ G₁ 714 I₁₂ 2218 N₄₄ H₁ R₁ D₁ 715 K₁₂ 2219 H₄₅ Y₁ R₁ 716 W₁₂ 2220 D₄₄ V₁ E₁ Y₁ 717 E₈ D₄ 2221 S₄₇ 718 Y₁₂ 2222 P₄₆ T₁ 719 V₁₂ 2223 D₄₇ 720 V₈ I₃ L₁ 2224 A₄₆ V₁ 721 L₁₂ 2225 E₄₅ D₂ 722 L₁₂ 2226 L₄₇ 723 F₁₂ 2227 I₄₇ 724 L₁₂ 2228 E₄₂ A₂ Q₂ T₁ 725 L₁₂ 2229 A₄₇ 726 L₁₂ 2230 N₄₇ 727 A₁₂ 2231 L₄₇ 728 D₁₂ 2232 L₄₇ 729 A₁₂ 2233 W₄₇ 730 R₁₂ 2234 R₄₆ N₁ 731 V₇ I₅ 2235 Q₄₇ 732 C₁₂ 2236 E₄₅ A₂ 733 S₁₂ 2237 M₄₇ 734 C₁₂ 2238 G₄₇ 735 L₁₂ 2239 G₄₆ C₁ 736 W₁₂ 2240 N₄₇ 737 M₁₂ 2241 I₄₇ 738 M₁₂ 2242 T₄₇ 739 L₁₂ 2243 R₄₇ 740 L₁₂ 2244 V₄₇ 741 I₁₂ 2245 E₄₇ 742 S₁₂ 2246 S₄₇ 743 Q₁₂ 2247 E₄₆ 744 A₁₁ V₁ 2248 N₄₆ S₁ 745 E₁₂ 2249 K₄₇ 746 A₁₂ 2250 V₄₇ 747 A₁₀ X₁ 2251 V₄₅ A₁ X₁ 748 L₁₀ 2252 I₄₀ V₆ 749 E₁₀ 2253 L₄₆ 750 N₁₀ 2254 D₄₆ 751 L₁₀ 2255 S₂₅ X₂₁ 752 V₉ I₁ 2256 F₄₆ 753 V₅ I₄ L₁ 2257 D₄₃ E₂ G₁ 754 L₁₀ 2258 P₄₆ 755 N₁₀ 2259 L₂₈ X₁₈ 756 A₁₀ 2260 V₂₄ X₁₈ C₂ R₁ T₁ 757 A₁₀ 2261 A₄₁ X₄ E₁ 758 S₁₀ 2262 E₂₅ X₂₀ 759 L₁₀ 2263 E₄₄ K₁ X₁ 760 A₁₀ 2264 D₄₆ 761 G₁₀ 2265 E₄₆ 762 T₁₀ 2266 R₄₅ Q₁ 763 H₈ R₁ Q₁ 2267 E₄₅ D₁ 764 G₁₀ 2268 X₂₁ I₁₄ V₁₁ 765 L₁₀ 2269 S₄₅ A₁ 766 V₇ A₃ 2270 V₄₃ T₁ A₁ I₁ 767 S₉ X₁ 2271 P₄₂ A₃ T₁ 768 F₉ 2272 A₄₆ 769 L₉ 2273 E₄₅ G₁ 770 V₈ M₁ 2274 I₄₅ M₁ 771 F₉ 2275 L₄₆ 772 F₉ 2276 R₄₅ L₁ 773 C₉ 2277 K₄₃ R₂ T₁ 774 F₈ L₁ 2278 S₄₂ Y₂ T₁ R₁ 775 A₉ 2279 R₄₅ G₁ 776 W₉ 2280 R₄₄ K₂ 777 Y₉ 2281 F₂₅ L₁₅ X₆ 778 L₉ 2282 A₂₇ X₁₁ P₄ T₄ 779 K₉ 2283 P₁₃ Q₁₂ X₁₀ R₇ S₂ A₁ E₁ 780 G₉ 2284 A₂₅ G₁₁ X₁₀ 781 R₅ K₄ 2285 L₄₆ 782 W₉ 2286 P₄₅ A₁ 783 V₉ 2287 V₂₅ I₂₁ 784 P₉ 2288 W₄₆ 785 G₉ 2289 A₂₅ X₂₁ 786 A₈ M₁ 2290 R₄₆ 787 A₅ V₄ 2291 P₄₆ 788 Y₉ 2292 D₄₄ E₂ 789 A₈ T₁ 2293 Y₄₆ 790 L₄ F₄ I₁ 2294 N₄₆ 791 Y₈ F₁ 2295 P₂₅ X₂₁ 792 G₉ 2296 P₄₆ 793 M₉ 2297 L₄₆ 794 W₉ 2298 L₂₃ V₁₂ I₁₀ M₁ 795 P₉ 2299 E₄₆ 796 L₈ F₁ 2300 T₃₉ A₄ S₁ L₁ E₁ 797 L₉ 2301 W₄₆ 798 L₉ 2302 K₃₂ X₁₄ 799 L₉ 2303 K₃₈ X₄ S₃ E₁ 800 L₉ 2304 P₄₃ X₃ 801 L₉ 2305 D₄₄ G₂ 802 A₉ 2306 Y₄₆ 803 L₉ 2307 E₄₄ K₂ 804 P₉ 2308 P₄₆ 805 Q₉ 2309 P₄₆ 806 R₉ 2310 V₄₅ T₁ 807 A₉ 2311 V₄₆ 808 Y₉ 2312 H₄₅ Y₁ 809 A₉ 2313 G₄₆ 810 L₉ 2314 C₃₄ X₁₂ 811 D₉ 2315 P₃₇ X₉ 812 T₉ 2316 L₄₆ 813 E₉ 2317 P₄₆ 814 V₇ M₂ 2318 P₄₅ S₁ 815 A₈ X₁ 2319 P₄₄ S₂ 816 A₈ 2320 Q₃₀ K₈ R₈ 817 S₈ 2321 S₄₅ P₁ 818 C₈ 2322 P₄₅ X₁ 819 G₈ 2323 P₂₅ X₂₀ S₁ 820 G₈ 2324 V₄₆ 821 V₈ 2325 P₄₆ 822 V₈ 2326 P₄₅ S₁ 823 L₈ 2327 P₄₆ 824 V₈ 2328 R₂₅ X₂₁ 825 G₈ 2329 K₃₉ R₇ 826 L₈ 2330 K₄₅ R₁ 827 M₈ 2331 R₄₆ 828 A₇ V₁ 2332 T₄₄ M₂ 829 L₈ 2333 V₄₅ I₁ 830 T₈ 2334 V₄₅ I₁ 831 L₈ 2335 L₄₆ 832 S₈ 2336 T₄₄ S₂ 833 P₈ 2337 E₄₆ 834 Y₇ H₁ 2338 S₄₆ 835 Y₈ 2339 T₃₆ S₄ N₃ P₁ A₁ L₁ 836 K₈ 2340 V₃₂ L₁₄ 837 R₇ H₁ 2341 S₄₃ P₃ 838 Y₈ 2342 T₄₂ S₂ A₂ 839 I₇ V₁ 2343 A₄₆ 840 S₈ 2344 L₄₆ 841 W₈ 2345 A₄₆ 842 C₈ 2346 E₄₅ 843 L₄ F₃ M₁ 2347 L₄₆ 844 W₈ 2348 A₄₆ 845 W₈ 2349 T₄₅ S₁ 846 L₈ 2350 K₄₄ R₁ I₁ 847 Q₈ 2351 S₃₁ X₁₃ T₂ 848 Y₈ 2352 F₃₈ X₈ 849 F₈ 2353 G₄₆ 850 L₈ 2354 S₄₄ D₁ G₁ 851 T₈ 2355 S₄₅ A₁ 852 R₈ 2356 S₄₆ 853 V₆ A₂ 2357 T₂₆ X₂₀ 854 E₈ 2358 S₄₅ X₁ 855 A₈ 2359 G₄₆ 856 Q₅ H₂ L₁ 2360 I₄₄ V₁ T₁ 857 L₈ 2361 T₄₀ A₃ S₂ V₁ 858 H₇ Q₁ 2362 G₃₈ S₅ A₃ 859 V₈ 2363 D₄₂ G₄ 860 W₈ 2364 N₄₀ D₄ S₂ 861 V₆ I₁ F₁ 2365 T₄₅ A₁ 862 P₈ 2366 T₄₁ A₄ P₁ 863 P₈ 2367 T₃₇ A₆ S₂ V₁ 864 L₈ 2368 S₃₈ P₈ 865 N₈ 2369 S₄₃ T₁ H₁ P₁ 866 V₇ A₁ 2370 E₄₆ 867 R₈ 2371 P₃₈ A₅ L₂ S₁ 868 G₈ 2372 A₃₇ T₅ S₃ P₁ 869 G₈ 2373 P₃₁ S₁₅ 870 R₈ 2374 S₃₉ P₆ T₁ 871 D₈ 2375 G₃₂ V₈ D₅ I₁ 872 A₈ 2376 C₃₉ R₄ X₂ H₁ 873 V₇ I₁ 2377 P₂₃ X₁₄ S₅ L₃ F₁ 874 I₈ 2378 P₄₁ X₅ 875 L₈ 2379 D₄₆ 876 L₈ 2380 S₄₆ 877 M₈ 2381 D₄₆ 878 C₈ 2382 A₃₆ V₅ T₃ D₁ N₁ 879 A₄ V₄ 2383 E₂₄ D₂₁ G₁ 880 V₆ I₂ 2384 S₂₅ X₂₁ 881 H₈ 2385 Y₃₂ C₁₂ F₁ N₁ 882 P₈ 2386 S₄₆ 883 T₆ A₁ S₁ 2387 S₄₆ 884 L₈ 2388 M₄₆ 885 V₈ 2389 P₂₇ X₁₉ 886 F₈ 2390 P₄₅ X₁ 887 D₈ 2391 L₄₅ X₁ 888 I₈ 2392 E₄₆ 889 T₈ 2393 G₄₆ 890 K₈ 2394 E₄₆ 891 L₈ 2395 P₄₆ 892 L₈ 2396 G₄₆ 893 L₈ 2397 D₄₅ G₁ 894 A₈ 2398 P₄₆ 895 I₄ V₃ A₁ 2399 D₄₆ 896 F₇ L₁ 2400 L₄₆ 897 G₈ 2401 S₄₆ 898 P₈ 2402 D₄₄ G₁ E₁ 899 L₈ 2403 G₄₅ A₁ 900 W₈ 2404 S₄₆ 901 I₈ 2405 W₄₆ 902 L₈ 2406 S₄₆ 903 Q₈ 2407 T₄₆ 904 A₆ T₂ 2408 V₄₆ 905 S₈ 2409 S₄₅ G₁ 906 L₈ 2410 S₄₅ N₁ 907 L₈ 2411 E₂₅ G₁₉ R₂ 908 K₈ 2412 A₄₁ T₂ D₁ P₁ S₁ 909 V₈ 2413 G₂₃ D₂₂ N₁ 910 P₈ 2414 T₃₇ A₈ G₁ 911 Y₈ 2415 E₄₆ X₂ 912 F₈ 2416 D₄₇ G₁ X₁ 913 V₈ 2417 V₅₀ 914 R₈ 2418 V₄₉ L₁ 915 V₈ 2419 C₅₀ 916 Q₈ 2420 C₂₉ 917 G₈ 2421 S₁₂ 918 L₈ 2422 M₁₂ 919 L₇ I₁ 2423 S₁₂ 920 R₈ 2424 Y₁₂ 921 I₆ F₁ V₁ 2425 S₁₁ T₁ 922 C₈ 2426 W₁₂ 923 A₇ V₁ 2427 T₁₂ 924 L₆ A₁ I₁ 2428 G₁₂ 925 A₇ S₁ 2429 A₁₂ 926 R₇ P₁ 2430 L₁₂ 927 K₈ 2431 V₇ I₅ 928 M₇ I₁ 2432 T₁₂ 929 A₅ V₂ I₁ 2433 P₁₂ 930 G₈ 2434 C₁₂ 931 G₇ L₁ 2435 A₁₁ T₁ 932 H₇ R₁ 2436 A₁₂ 933 Y₈ 2437 E₁₂ 934 V₈ 2438 E₁₁ G₁ 935 Q₈ 2439 Q₁₂ 936 M₈ 2440 K₁₂ 937 A₆ V₂ 2441 L₁₂ 938 I₆ M₁ V₁ 2442 P₁₂ 939 I₈ 2443 I₁₂ 940 K₈ 2444 N₁₂ 941 L₅ V₁ A₁ I₁ 2445 A₁₂ 942 G₈ 2446 L₁₂ 943 A₈ 2447 S₁₂ 944 L₈ 2448 N₁₂ 945 T₈ 2449 S₁₂ 946 G₈ 2450 L₁₂ 947 T₈ 2451 L₁₂ 948 Y₈ 2452 R₁₂ 949 V₆ I₂ 2453 H₁₂ 950 Y₈ 2454 H₁₂ 951 N₇ D₁ 2455 N₁₁ X₁ 952 H₈ 2456 L₁₂ 953 L₈ 2457 V₁₂ 954 T₈ 2458 Y₁₂ 955 P₈ 2459 S₁₂ 956 L₈ 2460 T₁₂ 957 R₈ 2461 T₁₂ 958 D₈ 2462 S₁₂ 959 W₈ 2463 R₁₂ 960 A₈ 2464 S₁₂ 961 H₈ 2465 A₁₂ 962 N₇ S₁ 2466 C₁₁ G₁ 963 G₇ S₁ 2467 Q₁₁ L₁ 964 L₈ 2468 R₁₂ 965 R₈ 2469 Q₁₂ 966 D₈ 2470 K₁₂ 967 L₈ 2471 K₁₂ 968 A₈ 2472 V₁₂ 969 V₈ 2473 T₁₂ 970 A₈ 2474 F₁₂ 971 V₈ 2475 D₁₂ 972 E₈ 2476 R₁₂ 973 P₈ 2477 L₁₁ V₁ 974 V₈ 2478 Q₁₂ 975 V₈ 2479 V₁₂ 976 F₈ 2480 L₁₂ 977 S₈ 2481 D₁₂ 978 Q₅ R₃ 2482 S₁₀ N₂ 979 M₈ 2483 H₁₂ 980 E₈ 2484 Y₁₂ 981 T₈ 2485 Q₁₁ R₁ 982 K₈ 2486 D₁₂ 983 L₈ 2487 V₁₂ 984 I₈ 2488 L₁₂ 985 T₈ 2489 K₁₂ 986 W₈ 2490 E₁₂ 987 G₈ 2491 V₁₂ 988 A₇ G₁ 2492 K₁₂ 989 D₈ 2493 A₁₂ 990 T₈ 2494 A₁₂ 991 A₈ 2495 A₁₂ 992 A₈ 2496 S₁₂ 993 C₈ 2497 K₁₂ 994 G₈ 2498 V₁₂ 995 D₈ 2499 K₁₂ 996 I₈ 2500 A₁₂ 997 I₈ 2501 N₁₂ 998 N₇ D₁ 2502 L₁₂ 999 G₇ N₁ 2503 L₁₂ 1000 L₈ 2504 S₁₂ 1001 P₈ 2505 V₁₂ 1002 V₈ 2506 E₁₂ 1003 S₈ 2507 E₁₂ 1004 A₈ 2508 A₁₂ 1005 R₈ 2509 C₁₂ 1006 R₈ 2510 S₁₂ 1007 G₈ 2511 L₁₂ 1008 R₆ Q₂ 2512 T₁₂ 1009 E₈ 2513 P₁₂ 1010 I₈ 2514 P₁₂ 1011 L₈ 2515 H₁₂ 1012 L₈ 2516 S₁₂ 1013 G₈ 2517 A₁₂ 1014 P₈ 2518 K₈ R₄ 1015 A₈ 2519 S₁₂ 1016 D₈ 2520 K₁₂ 1017 G₈ 2521 F₁₂ 1018 M₈ 2522 G₁₂ 1019 V₇ A₁ 2523 Y₁₂ 1020 S₈ 2524 G₁₂ 1021 K₈ 2525 A₁₂ 1022 G₈ 2526 K₁₂ 1023 W₈ 2527 D₁₄ 1024 R₈ 2528 V₁₄ 1025 L₈ 2529 R₁₄ 1026 L₇ Q₁ 2530 C₁₄ 1027 A₁₂₂ X₂ 2531 H₁₄ 1028 P₁₂₄ 2532 A₁₄ 1029 I₁₂₄ 2533 R₁₄ 1030 T₁₂₄ 2534 K₁₄ 1031 A₁₂₃ X₁ 2535 A₁₄ 1032 Y₁₂₄ 2536 V₁₄ 1033 A₁₂₂ T₂ 2537 N₈ A₅ T₁ 1034 Q₁₂₃ X₁ 2538 H₁₄ 1035 Q₁₂₄ 2539 I₁₄ 1036 T₁₂₄ 2540 N₁₄ 1037 R₁₂₂ X₂ 2541 S₁₄ 1038 G₁₂₄ 2542 V₁₄ 1039 L₁₂₄ 2543 W₁₄ 1040 L₁₂₂ X₂ 2544 K₁₄ 1041 G₁₂₃ R₁ 2545 D₁₄ 1042 C₁₂₃ X₁ 2546 L₁₄ 1043 I₁₂₄ 2547 L₁₄ 1044 I₁₀₈ V₁₄ X₂ 2548 E₁₄ 1045 T₁₂₄ 2549 D₁₃ A₁ 1046 S₁₂₃ N₁ 2550 S₁₁ N₃ 1047 L₁₂₄ 2551 V₁₄ 1048 T₁₂₄ 2552 T₁₄ 1049 G₁₂₄ 2553 P₁₄ 1050 R₁₂₄ 2554 I₁₄ 1051 D₁₂₄ 2555 D₁₄ 1052 K₁₂₂ R₁ X₁ 2556 T₁₄ 1053 N₁₂₃ D₁ 2557 T₁₄ 1054 Q₁₂₄ 2558 I₁₄ 1055 V₁₀₂ A₂₂ 2559 M₁₄ 1056 E₁₂₄ 2560 A₁₄ 1057 G₁₂₄ 2561 K₁₄ 1058 E₁₂₄ 2562 N₁₄ 1059 V₁₂₀ I₃ A₁ 2563 E₁₄ 1060 Q₁₂₂ H₁ X₁ 2564 V₁₄ 1061 I₁₂₂ X₁ V₁ 2565 F₁₄ 1062 V₁₂₄ 2566 C₁₄ 1063 S₁₂₄ 2567 V₁₄ 1064 T₁₂₄ 2568 Q₁₄ 1065 A₁₂₃ T₁ 2569 P₁₄ 1066 A₁₀₀ T₂₁ X₃ 2570 E₁₄ 1067 Q₁₂₄ 2571 K₁₄ 1068 T₁₂₂ S₁ X₁ 2572 G₁₄ 1069 F₁₂₃ X₁ 2573 G₁₄ 1070 L₁₂₄ 2574 R₁₃ C₁ 1071 A₁₂₃ X₁ 2575 K₁₄ 1072 T₁₂₀ X₄ 2576 P₁₄ 1073 C₁₂₃ S₁ 2577 A₁₄ 1074 I₁₂₄ 2578 R₁₄ 1075 N₁₂₄ 2579 L₁₄ 1076 G₁₂₄ 2580 I₁₄ 1077 V₁₂₃ X₁ 2581 V₁₄ 1078 C₁₂₄ 2582 F₁₃ Y₁ 1079 W₁₂₄ 2583 P₁₄ 1080 T₁₂₄ 2584 D₁₄ 1081 V₁₂₄ 2585 L₁₄ 1082 Y₁₂₄ 2586 G₁₄ 1083 H₁₂₄ 2587 V₁₄ 1084 G₁₂₄ 2588 R₁₄ 1085 A₁₂₃ X₁ 2589 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2801 V₁₀₂ 1298 K₄₅ 2802 Y₁₀₂ 1299 F₄₅ 2803 Y₁₀₂ 1300 L₄₅ 2804 L₁₀₁ X₁ 1301 A₄₅ 2805 T₁₀₂ 1302 D₄₄ X₁ 2806 R₁₀₂ 1303 G₄₃ X₂ 2807 D₁₀₂ 1304 G₄₅ 2808 P₁₀₁ X₁ 1305 C₄₄ X₁ 2809 T₁₀₁ A₁ 1306 S₄₅ 2810 T₁₀₂ 1307 G₄₅ 2811 P₁₀₂ 1308 G₄₄ X₁ 2812 L₉₇ F₄ I₁ 1309 A₄₅ 2813 A₁₀₀ V₁ X₁ 1310 Y₄₅ 2814 R₁₀₂ 1311 D₄₅ 2815 A₁₀₂ 1312 I₄₅ 2816 A₁₀₂ 1313 I₄₅ 2817 W₁₀₂ 1314 I₄₄ X₁ 2818 E₁₀₀ X₂ 1315 C₄₄ X₁ 2819 T₁₀₂ 1316 D₄₅ 2820 A₁₀₂ 1317 E₄₅ 2821 R₁₀₀ K₂ 1318 C₄₅ 2822 H₁₀₁ R₁ 1319 H₄₃ X₂ 2823 T₁₀₂ 1320 S₄₅ 2824 P₁₀₂ 1321 T₄₄ P₁ 2825 V₈₈ I₁₄ 1322 D₄₅ 2826 N₁₀₂ 1323 A₄₃ X₁ 2827 S₁₀₂ 1324 T₄₃ X₁ P₁ 2828 W₁₀₂ 1325 S₄₄ X₁ 2829 L₁₀₂ 1326 I₃₉ V₅ X₁ 2830 G₁₀₂ 1327 L₄₅ 2831 N₁₀₁ X₁ 1328 G₄₅ 2832 I₁₀₁ V₁ 1329 I₄₅ 2833 I₁₀₂ 1330 G₄₅ 2834 M₁₀₂ 1331 T₄₅ 2835 F₁₀₁ Y₁ 1332 V₄₃ A₂ 2836 A₁₀₂ 1333 L₄₂ X₃ 2837 P₁₀₂ 1334 D₄₅ 2838 T₁₀₂ 1335 Q₄₅ 2839 L₁₀₂ 1336 A₄₃ X₂ 2840 W₁₀₂ 1337 E₄₃ D₁ X₁ 2841 A₇₉ V₂₃ 1338 T₄₃ X₂ 2842 R₁₀₁ K₁ 1339 A₄₄ X₁ 2843 M₁₀₂ 1340 G₄₅ 2844 I₉₇ V₅ 1341 A₄₅ 2845 L₉₅ M₇ 1342 R₄₃ T₁ X₁ 2846 M₈₆ L₁₄ A₁ T₁ 1343 L₄₅ 2847 T₁₀₂ 1344 V₄₄ L₁ 2848 H₁₀₂ 1345 V₄₅ 2849 F₁₀₂ 1346 L₄₅ 2850 F₁₀₂ 1347 A₄₅ 2851 S₁₀₁ G₁ 1348 T₄₅ 2852 V₆₈ I₃₃ X₁ 1349 A₄₄ P₁ 2853 L₁₀₂ 1350 T₄₄ P₁ 2854 I₈₁ M₁₉ L₂ 1351 P₄₅ 2855 A₁₀₀ T₁ X₁ 1352 P₄₅ 2856 R₁₀₂ 1353 G₄₅ 2857 D₁₀₁ X₁ 1354 S₄₄ X₁ 2858 Q₁₀₂ 1355 V₃₆ I₇ X₂ 2859 L₁₀₂ 1356 T₄₃ S₁ X₁ 2860 E₁₀₂ 1357 V₄₄ X₁ 2861 Q₁₀₂ 1358 P₄₀ S₅ 2862 A₁₀₂ 1359 H₄₄ X₁ 2863 L₁₀₁ X₁ 1360 P₄₄ S₁ 2864 D₉₈ N₃ X₁ 1361 N₄₅ 2865 C₁₀₀ G₁ 1362 I₄₅ 2866 E₁₀₁ 1363 E₄₅ 2867 I₁₀₁ 1364 E₄₅ 2868 Y₁₀₀ 1365 V₄₃ A₁ I₁ 2869 G₁₀₁ 1366 A₄₅ 2870 A₉₇ X₂ 1367 L₄₅ 2871 C₉₂ X₃ 1368 S₄₃ P₂ 2872 Y₉₂ 1369 T₄₄ X₁ 2873 S₉₁ X₁ 1370 T₄₅ 2874 I₉₂ 1371 G₄₅ 2875 E₉₁ X₁ 1372 E₄₅ 2876 P₉₂ 1373 I₄₃ V₂ 2877 L₉₁ X₁ 1374 P₄₄ X₁ 2878 D₈₈ X₄ 1375 F₄₅ 2879 L₉₁ I₁ 1376 Y₄₅ 2880 P₈₇ X₅ 1377 G₄₅ 2881 P₇₃ X₆ S₃ Q₃ L₂ A₁ 1378 K₄₅ 2882 I₇₉ X₃ V₁ 1379 A₄₅ 2883 I₈₀ X₂ 1380 I₄₅ 2884 Q₈₀ X₂ 1381 P₄₅ 2885 X₆₁ R₆ 1382 L₄₅ 2886 L₆ 1383 E₄₄ G₁ 2887 H₅ 1384 V₃₂ A₁₁ X₂ 2888 G₅ 1385 I₄₄ X₁ 2889 L₅ 1386 K₄₃ R₁ X₁ 2890 S₅ 1387 G₄₅ 2891 A₅ 1388 G₄₅ 2892 F₅ 1389 R₄₅ 2893 S₅ 1390 H₄₅ 2894 L₅ 1391 L₄₅ 2895 H₅ 1392 I₄₅ 2896 S₅ 1393 F₄₅ 2897 Y₅ 1394 C₄₄ X₁ 2898 S₅ 1395 H₄₅ 2899 P₅ 1396 S₄₅ 2900 G₅ 1397 K₄₄ R₁ 2901 E₅ 1398 K₄₄ R₁ 2902 I₅ 1399 K₄₅ 2903 N₅ 1400 C₄₃ X₂ 2904 R₅ 1401 D₄₁ N₂ X₂ 2905 V₅ 1402 E₄₃ D₂ 2906 A₅ 1403 L₄₅ 2907 A₅ 1404 A₄₅ 2908 C₅ 1405 A₄₂ T₂ X₁ 2909 L₄ X₁ 1406 K₄₄ X₁ 2910 R₄ 1407 L₄₅ 2911 K₄ 1408 V₄₂ X₃ 2912 L₄ 1409 A₄₂ V₁ G₁ X₁ 2913 G₄ 1410 L₄₃ M₂ 2914 V₄ 1411 G₄₄ X₁ 2915 P₄ 1412 I₂₈ V₁₆ L₁ 2916 P₃ A₁ 1413 N₄₅ 2917 L₄ 1414 A₄₅ 2918 R₄ 1415 V₄₅ 2919 A₄ 1416 A₄₄ X₁ 2920 W₄ 1417 Y₄₃ X₂ 2921 R₄ 1418 Y₄₅ 2922 H₄ 1419 R₄₅ 2923 R₄ 1420 G₄₅ 2924 A₄ 1421 L₄₅ 2925 R₄ 1422 D₄₅ 2926 S₄ 1423 V₄₅ 2927 V₄ 1424 S₄₅ 2928 R₄ 1425 V₄₄ A₁ 2929 A₄ 1426 I₄₅ 2930 R₃ K₁ 1427 P₄₅ 2931 L₄ 1428 T₄₃ A₂ 2932 L₄ 1429 S₄₄ N₁ 2933 S₄ 1430 G₄₄ X₁ 2934 R₄ 1431 D₄₄ X₁ 2935 G₃ V₁ 1432 V₄₅ 2936 G₄ 1433 V₄₅ 2937 R₄ 1434 V₄₅ 2938 A₄ 1435 V₄₅ 2939 A₄ 1436 A₃₉ S₄ X₂ 2940 I₄ 1437 T₄₅ 2941 C₄ 1438 D₄₅ 2942 G₄ 1439 A₄₄ X₁ 2943 K₄ 1440 L₄₅ 2944 Y₄ 1441 M₄₅ 2945 L₄ 1442 T₄₃ X₂ 2946 F₄ 1443 G₄₅ 2947 N₄ 1444 Y₂₆ F₁₉ 2948 W₄ 1445 T₄₄ X₁ 2949 A₄ 1446 G₄₅ 2950 V₄ 1447 D₄₅ 2951 R₄ 1448 F₄₄ X₁ 2952 T₄ 1449 D₄₅ X₁ 2953 K₄ 1450 S₄₆ 2954 L₄ 1451 V₄₆ 2955 K₄ 1452 I₄₆ 2956 L₄ 1453 D₄₆ 2957 T₄ 1454 C₄₆ X₂ 2958 P₄ 1455 N₄₇ X₁ 2959 I₄ 1456 T₄₈ 2960 A₃ T₁ 1457 C₄₇ R₁ 2961 A₄ 1458 V₄₈ 2962 A₄ 1459 T₄₈ 2963 G₄ 1460 Q₄₇ X₂ 2964 Q₃ R₁ 1461 T₄₇ X₂ 2965 L₄ 1462 V₄₉ 2966 D₄ 1463 D₄₉ 2967 L₄ 1464 F₄₉ 2968 S₄ 1465 S₄₉ 2969 G₄ 1466 L₄₈ F₁ 2970 W₄ 1467 D₄₈ H₁ 2971 F₄ 1468 P₄₈ X₁ 2972 T₄ 1469 T₄₇ X₁ A₁ 2973 A₄ 1470 F₄₉ 2974 G₄ 1471 T₄₉ 2975 Y₄ 1472 I₄₈ X₁ 2976 S₄ 1473 E₄₆ D₃ 2977 G₄ 1474 T₄₈ I₁ 2978 G₄ 1475 T₄₆ S₂ I₁ 2979 D₄ 1476 T₄₉ 2980 I₄ 1477 L₄₈ X₁ 2981 Y₄ 1478 P₄₉ 2982 H₄ 1479 Q₄₈ X₁ 2983 S₄ 1480 D₄₉ 2984 V₄ 1481 A₄₇ X₂ 2985 S₄ 1482 V₄₇ X₂ 2986 R₃ H₁ 1483 S₄₉ 2987 A₄ 1484 R₄₆ X₃ 2988 R₄ 1485 T₄₈ S₁ 2989 P₄ 1486 Q₄₉ 2990 R₄ 1487 R₄₇ X₂ 2991 W₄ 1488 R₄₈ X₁ 2992 F₄ 1489 G₄₉ 2993 W₄ 1490 R₄₉ 2994 F₄ 1491 T₄₉ 2995 C₄ 1492 G₄₉ 2996 L₄ 1493 R₄₈ X₁ 2997 L₄ 1494 G₄₉ 2998 L₄ 1495 K₄₄ R₅ 2999 L₄ 1496 P₄₈ A₁ 3000 A₄ 1497 G₄₉ 3001 A₄ 1498 I₄₇ F₁ X₁ 3002 G₄ 1499 Y₄₉ 3003 V₄ 1500 R₄₉ 3004 G₃ C₁ 1501 F₄₉ 3005 I₄ 1502 V₄₉ 3006 Y₄ 1503 A₄₆ T₃ 3007 L₄ 1504 P₄₉ 3008 L₄ 1505 3009 P₄ 1506 3010 N₄ 1507 3011 R₄

TABLE 6 HCV 1b Consensus Sequences A B C D E F G H I J K L M N O P Q R 1 M₂₃₅ R₁ 2 S₂₃₅ Q₁ 3 T₂₃₅ E₁ 4 N₂₃₀ I₂ T₂ D₁ F₁ 5 P₂₃₄ L₁ G₁ 6 K₂₃₆ 7 P₂₃₆ 8 Q₂₃₆ 9 R₂₃₆ 10 K₂₂₇ Q₈ R₁ 11 T₂₃₂ I₃ S₁ 12 K₂₃₃ I₁ N₁ Y₁ 13 R₂₃₆ L₁ 14 N₂₃₆ 15 T₂₃₆ 16 N₂₂₉ Y₄ I₁ D₁ S₁ 17 R₂₃₄ L₂ 18 R₂₃₆ 19 P₂₃₆ 20 Q₂₃₆ 21 D₂₃₆ 22 V₂₃₃ I₂ L₁ 23 K₂₃₆ 24 F₂₃₆ 25 P₂₃₆ 26 G₂₃₅ A₁ 27 G₂₃₆ 28 G₂₃₆ 29 Q₂₃₄ K₁ R₁ 30 I₂₃₅ V₁ 31 V₂₃₆ 32 G₂₃₆ 33 G₂₃₆ 34 V₂₃₆ 35 Y₂₃₆ 36 L₂₃₅ V₁ 37 L₂₃₄ F₁ M₁ 38 P₂₃₄ T₂ 39 R₂₃₅ P₁ 40 R₂₃₆ 41 G₂₃₅ C₁ 42 P₂₃₆ 43 R₂₂₆ K₅ T₂ A₂ S₁ 44 L₂₃₆ 45 G₂₃₆ 46 V₂₃₆ 47 R₂₃₆ 48 A₂₃₃ T₁ P₁ X₁ 49 T₂₂₄ P₈ I₂ R₁ L₁ 50 R₂₃₆ 51 K₂₃₅ Q₁ 52 T₂₃₄ A₁ I₁ 53 S₂₃₅ W₁ 54 E₂₃₆ 55 R₂₃₆ 56 S₂₃₃ P₃ 57 Q₂₃₆ 58 P₂₃₄ A₂ 59 R₂₃₅ A₁ 60 G₂₃₅ E₁ 61 R₂₃₅ S₁ 62 R₂₃₅ P₁ 63 Q₂₃₅ H₁ 64 P₂₃₅ L₁ 65 I₂₃₅ V₁ 66 P₂₃₆ 67 K₂₃₃ R₁ N₁ E₁ 68 A₂₂₈ V₈ 69 R₂₃₆ 70 R₁₄₀ Q₉₀ H₄ K₁ X₁ 71 P₂₂₉ L₂ H₂ S₂ R₁ 72 E₂₃₆ 73 G₂₃₆ 74 R₂₃₅ W₁ 75 A₁₁₂ T₁₁₀ V₅ S₅ N₄ 76 W₂₃₄ C₁ S₁ 77 A₂₃₆ 78 Q₂₃₆ 79 P₂₃₆ 80 G₂₃₅ W₁ 81 Y₂₃₅ H₁ 82 P₂₃₆ 83 W₂₃₆ 84 P₂₃₆ 85 L₂₃₆ 86 Y₂₃₅ F₁ 87 G₂₃₁ A₅ 88 N₂₃₄ D₂ 89 E₂₃₅ Q₁ 90 G₂₃₅ S₁ 91 M₁₂₅ L₁₀₅ C₃ I₁ X₁ F₁ 92 G₂₃₆ 93 W₂₃₆ 94 A₂₃₃ T₂ P₁ 95 G₂₃₅ E₁ 96 W₂₃₅ L₁ 97 L₂₃₅ F₁ 98 L₂₃₅ V₁ 99 S₂₃₆ 100 P₂₃₆ 101 R₂₂₇ H₅ Q₂ Y₂ 102 G₂₃₅ S₁ 103 S₂₃₆ 104 R₂₃₆ 105 P₂₃₅ S₁ 106 S₂₁₆ N₁₈ R₂ 107 W₂₃₆ 108 G₂₃₆ 109 P₂₃₆ 110 T₂₀₅ N₁₆ S₁₂ R₁ K₁ I₁ 111 D₂₃₆ 112 P₂₃₆ 113 R₂₃₆ 114 R₂₃₄ C₂ 115 R₂₃₀ K₅ G₁ 116 S₂₃₆ X₁ 117 R₂₃₆ S₁ 118 N₂₃₆ I₁ 119 L₂₃₆ V₁ 120 G₂₃₇ 121 K₂₃₇ 122 V₂₃₆ I₁ 123 I₂₃₇ 124 D₂₃₇ 125 T₂₃₆ S₁ 126 L₂₃₆ F₁ P₁ 127 T₂₃₈ 128 C₂₃₈ 129 G₂₃₈ 130 F₂₂₇ L₈ V₃ 131 A₂₃₆ P₂ 132 D₂₃₈ 133 L₂₃₆ P₁ F₁ 134 M₂₃₆ V₁ L₁ 135 G₂₃₈ X₁₂ 136 Y₂₄₉ H₁ 137 I₂₄₆ V₂ L₂ 138 P₂₅₀ 139 L₂₄₂ R₅ P₁ F₁ V₁ 140 V₂₅₀ 141 G₂₅₀ 142 A₂₄₂ P₄ G₄ 143 P₂₅₀ 144 L₂₄₈ V₁ I₁ 145 G₂₄₇ R₂ A₁ 146 G₂₄₉ 147 A₂₀₂ V₄₈ 148 A₂₄₅ S₃ V₂ 149 R₂₄₉ K₁ 150 A₂₄₁ V₉ 151 L₂₅₀ X₁ 152 A₂₅₀ V₁ 153 H₂₅₁ 154 G₂₅₀ S₁ 155 V₂₅₁ X₁ 156 R₂₅₂ 157 V₂₄₆ A₅ X₃ I₂ L₁ 158 L₂₄₁ V₁₄ R₂ 159 E₂₅₅ L₂ 160 D₂₅₅ G₁ V₁ 161 G₂₃₃ S₂₄ 162 V₂₅₇ 163 N₂₅₇ 164 Y₂₅₇ 165 A₂₅₆ P₂ 166 T₂₅₈ X₁ 167 G₂₅₉ 168 N₂₅₉ X₁ 169 L₂₅₅ M₃ I₁ F₁ 170 P₂₅₉ A₁ 171 G₂₆₀ 172 C₂₆₀ 173 S₂₅₃ P₇ X₁ 174 F₂₆₁ 175 S₂₆₀ Y₁ 176 I₂₅₇ X₈ L₃ V₁ 177 F₂₇₂ S₃ 178 L₂₆₉ F₆ X₂ 179 L₂₆₈ W₃ S₂ M₂ G₁ I₁ 180 A₂₇₁ V₂ P₂ F₁ G₁ 181 L₂₇₃ F₃ V₁ 182 L₂₇₀ V₅ M₂ 183 S₂₇₇ 184 C₂₇₅ G₂ 185 L₂₇₅ V₁ M₁ 186 T₂₇₅ P1 I₁ 187 I₂₁₆ T₄₀ V₁₆ A₁ H₁ N₁ X₁ M₁ 188 P₂₇₅ Q₁ 189 A₂₄₆ V₂₁ T₇ N₁ G₁ 190 S₂₇₅ F₁ 191 A₂₇₄ T₂ G₁ 192 Y₁₈₂ I₃₁ H₉ X₆ P₁ 193 E₂₁₅ Q₅ D₂ V₁ K₁ 194 V₂₂₁ G₁ T₁ M₁ 195 R₂₀₈ H₉ G₄ A₁ N₁ C₁ 196 N₂₂₀ X₅ H₃ K₁ 197 V₁₈₈ A₃₅ X₁ I₁ L₁ G₁ T₁ S₁ 198 S₂₂₆ X₂ F₁ P₁ 199 G₂₂₆ R₂ D₁ E₁ 200 V₁₄₄ I₄₃ A₁₇ M₁₅ L₇ G₃ D₁ 201 Y₂₂₉ V₁ 202 H₂₂₁ Q₆ Y₂ L₁ 203 V₂₂₉ G₁ 204 T₂₂₉ S₁ 205 N₂₂₇ Y₁ H₁ S₁ 206 D₂₂₉ Y₁ 207 C₂₃₀ 208 S₂₂₈ P₂ 209 N₂₂₂ Y₃ S₂ H₁ K₁ Q₁ 210 S₁₇₉ A₄₄ T₆ L₁ 211 S₂₂₈ N₁ G₁ 212 I₂₂₉ V₁ 213 V₂₂₆ A₂ G₂ 214 Y₂₁₈ F₁₀ S₁ Q₁ 215 E₂₂₇ K₁ D₁ G₁ 216 A₁₉₃ T₃₃ V₃ P₁ 217 A₂₀₈ S₆ V₅ E₄ D₃ Q₂ T₁ K₁ 218 D₂₂₄ G₄ H₁ N₁ 219 M₁₆₁ V₃₅ L₁₉ I₁₂ T₂ S₁ 220 I₂₂₉ M₁ 221 M₂₁₅ L₁₄ I₁ 222 H₂₂₈ Q₁ D₁ 223 T₁₈₃ A₁₉ S₁₅ I₇ L₃ F₁ N₁ V₁ 224 P₂₃₀ 225 G₂₃₀ 226 C₂₂₉ F₁ 227 V₂₂₅ M₂ A₁ T₁ L₁ 228 P₂₂₉ A₁ 229 C₂₂₈ F₁ S₁ 230 V₂₃₀ 231 R₂₂₀ Q₄ L₂ W₂ S₁ G₁ 232 E₂₂₅ D₄ V₁ 233 N₁₀₆ G₄₉ D₄₀ S₁₆ A₁₁ K₃ E₂ Q₂ Y₁ 234 N₂₂₄ D₂ G₂ Y₁ K₁ 235 S₁₈₃ F₁₂ A₇ I₆ T₆ H₅ L₅ V₄ Y₁ C₁ 236 S₂₃₁ 237 R₂₂₄ S₃ H₂ K₁ Q₁ 238 C₂₂₉ Y₂ 239 W₂₃₁ 240 V₂₂₃ A₅ I₂ T₁ 241 A₂₃₀ E₁ 242 L₂₂₉ V₁ I₁ 243 T₂₂₅ A₆ 244 P₂₃₀ L₁ 245 T₂₃₁ 246 L₂₂₈ V₁ P₁ H₁ 247 A₂₂₉ S₂ 248 A₂₂₈ T₁ S₁ G₁ 249 R₂₂₈ K₃ 250 N₂₂₈ D₂ S₁ 251 S₉₂ A₈₅ T₁₄ G₉ I₉ N₇ V₅ R₃ F₃ D₁ H₁ L₁ P₁ 252 S₂₁₇ T₁₀ N₃ R₁ 253 V₁₃₂ I₉₈ L₁ 254 P₂₂₉ S₂ 255 T₂₂₈ V₂ I₁ 256 T₁₈₆ A₂₄ K₁₅ M₆ 257 T₁₉₇ A₂₉ S₄ Q₁ 258 I₂₁₂ L₁₀ M₅ V₄ 259 R₂₃₁ 260 R₂₂₅ H₄ D₁ C₁ 261 H₂₃₁ 262 V₂₂₆ I₅ 263 D₂₃₀ N₁ 264 L₂₃₀ V₁ 265 L₂₃₀ H₁ 266 V₂₃₁ 267 G₂₄₂ 268 A₁₉₀ T₄₇ V₄ G₁ 269 A₂₄₂ 270 A₂₂₂ T₁₆ V₄ 271 F₂₁₁ L₃₁ 272 C₂₂₈ S₇ W₅ L₁ R₁ 273 S₂₄₁ A₁ 274 A₂₃₅ V₆ S₁ 275 M₂₄₁ L₁ 276 Y₂₄₅ 277 V₂₄₃ G₁ M₁ 278 G₂₄₃ W₁ E₁ 279 D₂₄₄ N₁ 280 L₂₃₆ F₈ X₁ 281 C₂₄₅ 282 G₂₄₄ R₁ 283 S₂₄₅ 284 V₂₃₆ I₅ A₃ X₁ 285 F₂₃₂ L₁₃ 286 L₂₄₄ P₁ 287 V₂₁₅ I₂₇ A₂ L₁ 288 S₂₃₉ G₃ A₃ 289 Q₂₄₅ 290 L₂₄₅ 291 F₂₄₃ I₁ L₁ 292 T₂₄₁ V₃ A₁ 293 F₂₃₃ L₉ I₃ 294 S₂₄₄ L₁ 295 P₂₄₂ A₂ T₁ 296 R₂₄₃ S₁ P₁ 297 R₂₂₀ Q₁₄ W₃ L₃ H₂ V₂ G₁ 298 H₁₈₅ Y₆₀ 299 E₂₀₆ Q₉ M₆ V₆ W₄ T₃ A₃ N₂ G₂ K₂ R₂ 300 T₂₄₂ I₂ V₁ 301 V₂₁₆ I₈ L₇ T₇ A₅ E₂ 302 Q₂₄₃ H₁ R₁ 303 D₂₂₈ E₁₂ N₂ Y₁ T₁ S₁ 304 C₂₄₅ 305 N₂₄₃ K₁ D₁ 306 C₂₄₅ 307 S₂₄₅ 308 I₂₀₂ L₄₂ H₁ 309 Y₂₄₄ F₁ 310 P₂₃₈ L₃ S₂ A₁ V₁ 311 G₂₄₅ 312 H₂₄₁ R₃ K₁ 313 V₁₉₄ L₃₄ I₁₅ A₂ 314 S₁₆₄ T₈₁ 315 G₂₄₃ V₁ S₁ 316 H₂₄₄ Y₁ 317 R₂₄₄ A₁ 318 M₂₄₉ I₁ 319 A₂₅₀ X₅ 320 W₂₅₄ X₁₀ L₁ 321 D₂₆₁ X₂₄ N₃ E₁ 322 M₂₈₈ V₁ X₁ 323 M₂₈₆ I₃ R₂ X₁ 324 M₂₉₁ X₃ 325 N₂₉₀ T₁ 326 W₂₉₀ C₁ 327 S₂₉₁ 328 P₃₁₃ L₄ A₁ H₁ 329 T₃₁₅ A₅ K₂ Q₁ S₁ 330 T₂₀₉ A₁₁₅ S₃ X₂ I₁ Q₁ L₁ K₁ V₁ 331 A₃₂₉ G₃ T₃ L₂ D₁ S₁ P₁ V₁ 332 L₃₃₂ I₅ P₄ M₂ X₁ 333 V₃₃₄ I₄ M₂ G₂ S₁ L₁ 334 V₃₂₈ M₆ L₅ A₄ P₁ G₁ 335 S₃₂₆ A₉ V₃ L₂ C₁ X₁ G₁ H₁ D₁ 336 Q₃₃₈ W₂ H₂ Y₁ E₁ V₁ 337 L₃₂₄ I₆ X₆ V₅ M₁ Y₁ A₁ F₁ 338 L₃₃₁ F₃ P₂ M₁ G₁ 339 R₃₃₅ P₁ M₁ Q₁ 340 I₃₂₀ L₈ V₇ N₁ X₁ Y₁ 341 P₃₃₀ X₇ R₁ 342 Q₃₃₃ E₁ 343 A₃₂₅ T₆ S₂ V₁ 344 V₂₆₁ I₆₄ A₇ T₂ 345 V₂₃₁ M₇₆ L₂₄ I₃ G₁ 346 D₃₃₂ X₁ N₁ L₁ 347 M₃₁₃ I₁₀ V₁₀ T₁ E₁ 348 V₃₀₉ I₁₈ M₄ L₄ 349 A₃₀₁ V₁₄ T₁₃ G₆ S₁ 350 G₃₃₅ 351 A₃₂₆ G₆ S₂ X₁ 352 H₃₃₁ Y₁ Q₁ L₁ 353 W₃₃₄ 354 G₃₃₂ E₁ X₁ 355 V₃₁₆ I₁₇ A₁ 356 L₃₃₁ M₁ P₁ X₁ 357 A₃₂₉ V₄ F₁ 358 G₃₃₃ D₁ 359 L₃₁₅ I₁₈ P₁ 360 A₃₃₁ G₂ V₁ 361 Y₃₃₃ 362 Y₃₁₈ F₁₅ 363 S₃₂₂ A₇ T₂ P₂ 364 M₃₃₃ 365 V₂₉₀ A₃₈ I₆ Q₁ 366 G₃₃₀ A₄ R₁ 367 N₃₃₂ K₁ S₁ A₁ 368 W₃₃₅ 369 A₃₃₄ X₄ 370 K₃₃₈ 371 V₃₃₄ A₂ L₁ 372 L₃₃₁ V₄ M₁ F₁ 373 I₂₉₉ V₃₂ L₆ 374 V₃₂₉ X₅ A₂ I₁ 375 M₃₀₆ L₂₅ I₁ 376 L₃₃₂ 377 L₃₂₉ P₂ R₁ 378 F₃₃₁ A₁ 379 A₃₂₄ S₆ L₁ V₁ 380 G₃₃₀ S₁ R₁ 381 V₃₂₉ A₃ 382 D₃₂₉ N₂ A₁ 383 G₃₀₄ A₁₉ R₃ E₁ Q₁ T₁ L₁ P₁ X₁ 384 S₄₃ T₄₁ E₄₀ G₃₄ D₃₁ N₃₀ H₂₈ Q₂₁ R₂₀ A₁₆ K₄ V₄ Y₂ M₁ P₁ 385 T₃₁₁ D₁ S₁ N₁ I₁ A₁ 386 H₁₄₄ Y₆₉ R₅₉ T₁₄ V₆ S₅ L₅ N₃ Q₃ I₂ M₁ A₁ F₁ K₁ G₁ 387 V₁₈₁ T₁₀₇ A₁₂ I₅ L₄ M₃ Q₁ S₁ 388 T₁₈₃ S₅₆ V₂₆ I₂₁ M₁₄ L₇ A₅ W₁ X₁ 389 G₃₁₄ V₁ 390 G₂₇₈ A₂₇ E₆ S₂ V₁ K₁ 391 A₁₁₀ T₈₆ V₄₉ S₂₉ Q₂₀ E₆ K₅ H₄ R₃ X₁ N₁ M₁ 392 A₁₁₀ Q₁₀₉ V₃₁ T₂₈ S₁₄ E₆ P₄ L₃ N₂ H₂ M₂ K₁ Y₁ R₁ I₁ 393 A₁₄₃ G₁₃₀ S₄₀ T₁ X₁ 394 R₁₄₄ H₈₈ Y₂₈ Q₁₅ F₁₂ S₁₁ L₇ K₇ M₁ D₁ A₁ 395 T₁₄₇ N₄₈ S₄₁ A₃₇ G₁₁ V₆ D₅ H₄ Q₃ Y₃ L₃ M₁ R₁ I₁ K₁ E₁ C₁ F₁ 396 T₁₉₆ A₅₉ L₂₂ I₁₈ V₁₇ S₁ G₁ M₁ 397 R₈₉ S₈₅ Q₃₃ H₃₀ Y₂₅ L₁₇ N₁₀ F₇ A₆ G₄ W₃ V₂ K₁ M₁ T₁ I₁ 398 G₁₈₀ S₆₃ R₃₈ T₁₃ Q₆ K₅ A₃ V₂ I₂ L₁ M₁ E₁ 399 L₁₄₃ F₁₃₃ I₂₇ V₁₀ H₁ M₁ 400 T₁₈₀ A₉₄ V₃₄ M₃ S₃ R₁ 401 S₂₄₅ G₂₉ T₁₆ N₁₀ A₇ R₅ D₁ H₁ Y₁ 402 L₂₃₅ F₄₄ I₂₄ M₉ W₂ X₁ 403 F₂₉₃ L₂₁ Y₁ 404 S₁₃₃ T₁₁₉ A₂₈ N₁₄ R₉ Q₃ D₃ M₂ K₁ L₁ I₁ V₁ 405 P₁₃₅ S₅₈ L₃₃ R₂₃ V₁₈ F₁₇ A₁₀ Q₈ T₇ C₂ I₂ H₁ Y₁ 406 G₃₁₄ E₁ 407 P₁₇₃ A₁₀₃ S₃₆ Q₁ X₁ R₁ 408 S₁₉₆ A₆₁ K₁₆ Q₁₃ T₁₂ R₆ H₄ N₃ V₂ E₁ L₁ 409 Q₃₁₀ H₄ E₁ 410 K₁₉₃ N₈₅ R₂₆ T₃ D₃ E₂ H₂ P₁ 411 I₂₇₄ V₂₀ L₂₀ N₁ 412 Q₃₁₁ K₂ E₁ 413 L₃₁₂ I₂ 414 V₁₆₃ I₁₄₅ M₃ X₂ 415 N₃₀₃ K₅ H₂ Y₁ 416 T₂₉₁ S₁₅ N₂ A₁ 417 N₃₀₅ S₁ E₁ 418 G₃₀₅ D₁ X₁ 419 S₃₀₄ N₂ I₁ 420 W₃₀₄ R₁ 421 H₃₀₅ 422 I₂₇₄ V₃₁ 423 N₃₀₅ 424 R₂₉₈ S₇ 425 T₃₀₅ 426 A₃₀₃ S₁ X₁ 427 L₃₀₄ 428 N₂₉₈ S₅ D₁ 429 C₃₀₃ W₁ 430 N₂₈₉ D₁₀ K₃ Y₁ X₁ 431 D₂₈₃ A₁₁ E₈ G₁ X₁ 432 S₂₈₄ T₁₆ A₂ P₁ 433 L₂₉₂ I₄ F₃ H₃ Y₁ 434 Q₁₁₇ N₁₀₈ K₃₄ H₂₄ D₇ S₆ E₃ R₃ 435 T₂₉₅ S₁ A₁ 436 G₂₉₇ 437 F₂₇₇ W₂₀ 438 L₁₈₅ I₉₃ V₁₀ F₉ 439 A₂₈₉ T₄ S₃ G₁ 440 A₂₇₅ G₁₆ S₅ T₁ 441 L₂₉₆ V₁ 442 F₂₈₃ L₆ I₅ S₁ V₁ 443 Y₂₉₅ F₁ 444 T₁₅₉ A₈₃ V₂₅ Y₈ H₈ K₅ R₄ F₂ Q₁ L₁ 445 H₂₀₁ N₄₁ R₃₇ Y₉ K₆ S₂ 446 K₁₄₉ R₁₀₅ S₁₉ N₁₆ Q₃ E₂ M₁ G₁ 447 F₂₈₃ I₆ L₆ V₁ 448 N₂₉₄ K₁ D₁ 449 A₁₄₄ S₁₃₃ T₇ D₇ G₂ E₁ P₁ M₁ 450 S₂₈₇ T₆ C₁ A₁ P₁ 451 G₂₉₄ E₁ R₁ 452 C₂₉₃ X₂ G₁ 453 P₂₅₈ S₂₀ L₆ T₃ A₂ R₂ I₂ V₁ G₁ 454 E₂₇₀ Q₁₃ A₆ G₃ K₁ D₁ P₁ 455 R₂₈₇ K₃ H₂ G₂ P₁ 456 M₂₃₄ L₆₀ I₁ 457 A₂₈₃ S₅ X₃ V₂ N₁ T₁ 458 S₂₇₈ T₄ G₃ Q₂ R₁ H₁ V₁ N₁ X₁ 459 C₂₉₁ 460 R₂₇₂ S₆ K₆ H₃ Q₂ L₁ C₁ 461 P₂₀₆ S₇₅ T₃ R₂ N₁ A₁ X₁ F₁ H₁ 462 I₂₆₇ L₂₀ V₃ 463 D₂₅₃ A₁₁ S₆ T₆ N₅ E₄ G₃ V₁ 464 K₁₅₀ E₅₁ T₃₂ Q₁₇ A₁₀ D₉ R₇ G₅ N₃ S₂ Y₁ H₁ W₁ 465 F₂₈₈ Y₁ 466 A₁₄₉ D₈₇ S₂₅ N₁₀ V₅ T₅ E₃ H₃ G₁ R₁ 467 Q₂₈₈ I₁ 468 G₂₈₉ 469 W₂₈₇ F₁ S₁ 470 G₂₈₈ X₁ 471 P₂₇₅ S₁₂ A₁ L₁ 472 I₂₈₄ V₃ L₂ 473 T₂₆₆ S₁₃ A₃ N₂ R₂ Q₁ H₁ K₁ 474 Y₂₄₈ H₃₇ N₁ F₁ C₁ S₁ 475 A₁₆₅ V₃₈ T₃₆ D₂₈ G₁₁ N₆ I₂ H₁ S₁ Y₁ 476 E₁₅₃ K₃₃ V₂₄ G₁₅ N₁₅ Q₁₂ R₉ A₉ D₇ T₄ S₄ M₃ P₁ 477 P₁₇₅ S₄₈ G₂₆ A₈ D₆ Q₆ R₆ H₅ Y₃ T₂ N₂ L₁ V₁ 478 D₆₈ N₄₂ S₃₅ G₃₅ P₃₁ R₂₃ H₁₈ T₁₆ A₇ E₄ V₄ Q₃ K₂ L₁ 479 S₁₂₀ G₅₀ D₄₁ N₂₇ I₂₇ V₈ R₅ T₃ A₂ K₂ P₂ H₁ L₁ 480 S₁₁₆ L₇₀ P₅₈ Q₂₆ V₄ R₄ M₃ T₃ K₂ E₁ X₁ W₁ 481 D₂₇₁ E₁₃ X₃ G₁ 482 Q₂₆₉ H₁₄ R₁ M₁ 483 R₂₃₅ K₄₈ G₁ S₁ 484 P₂₈₄ L₁ 485 Y₂₈₅ 486 C₂₈₅ 487 W₂₈₃ S₁ G₁ 488 H₂₈₃ S₁ P₁ 489 Y₂₇₁ X₁₃ N₁ 490 A₂₃₀ P₃₀ R₁ 491 P₂₅₉ Q₁ L₁ 492 R₁₅₇ Q₆₂ K₃₆ P₃ E₂ S₁ 493 P₁₉₃ Q₄₀ R₁₀ K₉ L₆ A₁ X₁ S₁ 494 C₂₅₉ 495 G₂₄₉ S₃ D₂ K₁ V₁ X₁ N₁ T₁ 496 I₂₃₈ T₁₁ V₁₀ 497 V₂₃₃ I₂₂ X₁ A₁ T₁ E₁ 498 P₂₄₉ S₆ X₂ A₁ H₁ 499 A₂₄₈ P₂ X₂ T₂ W₁ 500 S₂₁₁ A₁₅ L₁₃ Q₇ R₃ K₂ X₁ 501 Q₁₃₃ E₇₄ K₂₀ T₇ N₇ G₃ D₃ R₁ S₁ 502 V₂₄₃ A₁ C₁ M₁ X₁ 503 C₂₂₈ X₁₄ P₁ 504 G₂₂₇ V₅ A₁ X₁ S₁ 505 P₂₂₈ Q₆ G₁ 506 V₂₂₃ X₈ M₃ 507 Y₁₈₃ H₁ X₁ 508 C₁₈₃ R₁ 509 F₁₇₉ L₁ X₁ 510 T₁₇₅ X₅ 511 P₁₇₄ 512 S₁₇₁ T₂ A₁ 513 P₁₇₄ 514 V₁₇₁ I₂ 515 V₁₇₁ A₂ 516 V₁₇₁ N₁ X₁ 517 G₁₇₁ W₁ 518 T₁₆₈ S₂ K₁ X₁ 519 T₁₇₁ S₁ 520 D₁₇₀ N₁ 521 R₁₆₁ H₅ S₂ K₁ G₁ P₁ 522 F₁₁₁ S₄₅ L₉ Y₂ H₂ A₁ T₁ 523 G₁₆₈ S₂ 524 V₁₁₂ A₅₅ N₂ T₁ 525 P₁₆₉ R₁ 526 T₁₇₀ 527 Y₁₅₈ X₁ S₁ 528 S₇₇ N₃₆ T₂₄ R₁₈ K₂ D₁ V₁ 529 W₁₅₈ X₁₀ R₁ 530 G₁₆₅ X₃ R₁ 531 E₁₃₁ A₁₆ D₆ G₅ V₄ N₃ S₁ 532 N₁₆₄ S₂ 533 E₁₅₈ D₃ K₂ G₂ V₁ 534 T₁₆₃ S₂ I₁ 535 D₁₆₆ 536 V₁₆₃ M₁ A₁ I₁ 537 L₁₆₅ F₁ 538 L₁₄₂ I₁₂ V₇ F₂ P₂ Y₁ 539 L₁₆₆ 540 N₁₆₄ T₁ S₁ 541 N₁₆₂ S₄ 542 T₁₅₇ S₄ M₂ A₂ X₁ 543 R₁₆₃ G₂ L₁ 544 P₁₆₃ L₂ A₁ 545 P₁₅₅ X₁₁ 546 Q₁₁₂ R₂₈ H₇ L₇ P₁ 547 G₁₅₃ A₁ D₁ 548 N₁₅₁ T₂ S₂ 549 W₁₅₄ K₁ 550 F₁₅₅ 551 G₁₅₅ 552 C₁₅₄ Y₁ 553 T₁₅₄ S₁ 554 W₁₅₄ Y₁ 555 M₁₅₃ I₁ V₁ 556 N₁₅₄ S₁ 557 S₉₉ G₄₄ A₈ T₃ N₁ 558 T₁₅₃ S₂ 559 G₁₅₄ R₁ 560 F₁₅₀ Y₄ S₁ 561 T₁₅₄ A₁ 562 K₁₅₄ M₁ 563 T₁₅₃ V₁ A₁ 564 C₁₅₅ 565 G₁₅₄ E₁ 566 G₁₄₂ A₁₀ R₁ D₁ V₁ 567 P₁₅₅ 568 P₁₅₁ S₂ A₁ L₁ 569 C₁₅₅ 570 N₁₃₀ D₉ K₇ G₃ H₂ T₂ V₁ E₁ 571 I₁₅₅ 572 G₁₅₂ E₁ S₁ R₁ 573 G₁₅₅ 574 V₁₀₅ A₃₃ I₆ G₅ E₂ L₂ N₁ R₁ 575 G₁₅₃ S₁ D₁ 576 N₁₅₃ S₁ 577 N₁₁₃ D₃₄ K₃ T₂ L₁ S₁ E₁ 578 T₁₅₀ S₃ R₁ A₁ 579 L₁₅₄ F₁ 580 T₈₅ I₅₈ V₁₀ H₁ L₁ 581 C₁₅₅ 582 P₁₅₃ S₂ 583 T₁₅₄ S₁ 584 D₁₅₅ 585 C₁₅₅ 586 F₁₅₅ 587 R₁₅₅ 588 K₁₅₃ X₁ E₁ 589 H₁₅₄ 590 P₁₅₃ S₁ 591 E₁₄₅ G₃ D₃ A₂ Q₁ 592 A₁₅₃ S₁ 593 T₁₅₃ A₁ 594 Y₁₅₄ 595 T₁₄₁ A₉ S₃ X₁ 596 K₁₄₄ R₁₀ 597 C₁₅₄ 598 G₁₅₃ X₁ 599 S₁₅₄ 600 G₁₅₄ 601 P₁₅₄ 602 W₁₅₄ 603 L₁₅₂ S₁ I₁ 604 T₁₄₉ R₁ I₁ 605 P₁₅₁ 606 R₁₅₀ K₁ 607 C₁₅₁ 608 M₆₉ L₅₁ I₃₁ 609 V₁₄₉ I₂ 610 D₁₂₆ H₂₄ N₁ 611 Y₁₅₁ 612 P₁₅₁ 613 Y₁₅₁ 614 R₁₅₁ 615 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1414 A₁₃₃ 1415 V₁₃₂ I₁ 1416 A₁₃₁ V₁ S₁ 1417 Y₁₃₃ 1418 Y₁₃₃ 1419 R₁₃₃ 1420 G₁₃₃ 1421 L₁₃₁ F₂ 1422 D₁₃₃ 1423 V₁₃₃ 1424 S₁₃₃ 1425 V₁₂₇ I₅ D₁ 1426 I₁₃₃ 1427 P₁₃₃ 1428 T₁₁₅ A₁₀ S₆ V₁ P₁ 1429 S₁₂₈ N₄ I₁ 1430 G₁₃₃ 1431 D₁₂₀ N₁₃ 1432 V₁₃₀ X₁ D₁ A₁ 1433 V₁₃₀ I₃ 1434 V₁₃₁ A₁ I₁ 1435 V₁₃₃ 1436 A₁₃₂ T₁ 1437 T₁₃₂ S₁ 1438 D₁₃₃ 1439 A₁₃₃ 1440 L₁₃₃ 1441 M₁₃₃ 1442 T₁₃₃ 1443 G₁₃₄ 1444 F₇₈ Y₅₆ 1445 T₁₃₄ 1446 G₁₃₄ 1447 D₁₃₂ N₂ 1448 F₁₃₂ L₁ S₁ 1449 D₁₃₃ X₁ V₁ 1450 S₁₃₅ 1451 V₁₃₃ W₁ X₁ 1452 I₁₃₄ T₁ 1453 D₁₃₅ 1454 C₁₃₄ R₁ 1455 N₁₃₅ 1456 T₁₃₁ V₃ X₁ 1457 C₁₃₄ X₁ 1458 V₁₃₅ 1459 T₁₃₀ I₃ N₁ A₁ 1460 Q₁₃₅ 1461 T₁₃₅ 1462 V₁₃₅ 1463 D₁₃₅ 1464 F₁₃₄ L₁ 1465 S₁₃₅ 1466 L₁₃₄ 1467 D₁₃₄ 1468 P₁₃₄ 1469 T₁₃₄ 1470 F₁₃₄ 1471 T₁₃₄ 1472 I₁₃₃ M₁ 1473 E₁₃₁ D₃ 1474 T₁₃₃ X₁ 1475 T₁₃₁ M₁ A₁ 1476 T₁₃₃ 1477 V₁₂₈ L₃ M₂ 1478 P₁₃₃ 1479 Q₁₃₂ H₁ 1480 D₁₃₂ Y₁ 1481 A₁₃₁ S₂ 1482 V₁₃₃ 1483 S₁₃₂ A₁ 1484 R₁₃₁ S₁ A₁ 1485 S₁₂₃ T₈ A₂ 1486 Q₁₃₃ 1487 R₁₃₁ L₁ X₁ 1488 R₁₃₃ 1489 G₁₃₃ 1490 R₁₃₂ K₁ 1491 T₁₃₃ 1492 G₁₃₂ S₁ 1493 R₁₃₂ S₁ 1494 G₁₃₃ 1495 R₁₃₂ T₁ 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T₁₂₈ S₂ 1741 K₁₂₈ T₁ N₁ 1742 Q₁₃₀ 1743 A₁₃₀ 1744 E₁₂₂ X₈ 1745 A₁₁₈ V₄ 1746 A₁₂₁ V₁ 1747 A₁₁₈ V₃ X₁ 1748 P₁₂₂ 1749 V₁₁₇ A₃ I₁ M₁ 1750 V₁₂₁ M₁ 1751 E₁₂₂ 1752 S₁₂₁ T₁ 1753 K₁₁₈ R₄ 1754 W₁₂₂ 1755 R₁₁₂ Q₈ K₂ 1756 A₁₀₄ T₁₆ G₂ 1757 L₁₂₂ 1758 E₁₂₂ 1759 T₄₄ A₄₁ S₃₁ V₄ G₂ 1760 F₁₂₂ 1761 W₁₂₂ 1762 A₁₂₀ E₁ G₁ 1763 K₁₂₀ N₂ 1764 H₁₂₀ D₂ 1765 M₁₂₀ X₂ 1766 W₁₁₉ R₁ 1767 N₁₁₉ S₁ 1768 F₁₂₀ 1769 I₁₂₀ 1770 S₁₂₀ 1771 G₁₂₀ 1772 I₁₁₄ V₆ 1773 Q₁₂₀ 1774 Y₁₂₀ 1775 L₁₂₀ 1776 A₁₂₀ 1777 G₁₁₉ A₁ 1778 L₁₂₀ 1779 S₁₂₀ 1780 T₁₂₀ 1781 L₁₂₀ 1782 P₁₁₉ H₁ 1783 G₁₂₀ 1784 N₁₂₀ 1785 P₁₁₉ L₁ 1786 A₁₂₀ 1787 I₁₁₉ M₁ 1788 A₁₁₈ R₂ 1789 S₁₂₀ 1790 L₁₁₈ P₂ 1791 M₁₂₀ 1792 A₁₁₈ E₁ V₁ 1793 F₁₂₀ 1794 T₁₂₀ 1795 A₁₁₉ S₁ 1796 S₁₁₉ A₁ 1797 I₁₀₈ V₁₂ 1798 T₁₂₀ 1799 S₁₁₉ X₁ 1800 P₁₁₉ 1801 L₁₁₆ F₃ 1802 T₁₁₇ S₁ A₁ 1803 T₁₁₈ I₁ 1804 Q₁₁₇ N₂ 1805 H₅₇ S₃₀ N₂₂ Y₇ Q₂ T₁ 1806 T₁₁₉ 1807 L₁₁₈ F₁ 1808 L₁₁₇ M₂ 1809 F₁₁₈ L₁ 1810 N₁₁₈ D₁ 1811 I₁₁₉ 1812 L₁₁₈ W₁ 1813 G₁₁₉ 1814 G₁₁₉ 1815 W₁₁₉ 1816 V₁₁₈ L₁ 1817 A₁₁₉ 1818 A₁₁₉ 1819 Q₁₁₈ X₁ 1820 L₁₁₇ I₁ P₁ 1821 A₁₁₈ P₁ 1822 P₁₁₉ 1823 P₁₁₈ A₁ 1824 S₁₁₁ G₄ R₃ N₁ 1825 A₁₁₉ 1826 A₁₁₇ S₁ V₁ 1827 S₁₁₈ T₁ 1828 A₁₁₉ 1829 F₁₁₉ 1830 V₁₁₉ 1831 G₁₁₉ 1832 A₁₁₉ 1833 G₁₁₉ 1834 I₁₁₇ S₁ V₁ 1835 A₁₁₂ V₅ T₂ 1836 G₁₁₇ X₁ R₁ 1837 A₁₁₈ X₁ 1838 A₁₁₉ 1839 V₁₀₉ I₁₀ 1840 G₁₁₉ 1841 S₁₁₆ T₃ 1842 I₁₁₆ V₃ 1843 G₁₁₉ 1844 L₁₁₇ V₁ F₁ 1845 G₁₁₉ 1846 K₁₁₆ R₃ 1847 V₁₁₉ 1848 L₁₁₇ I₂ 1849 V₁₁₉ 1850 D₁₁₈ E₁ 1851 I₁₁₅ V₃ M₁ 1852 L₁₁₈ V₁ 1853 A₁₁₉ 1854 G₁₁₉ 1855 Y₁₁₉ 1856 G₁₁₉ 1857 A₁₁₉ 1858 G₁₁₉ 1859 V₁₁₈ X₁ 1860 A₁₁₉ 1861 G₁₁₉ 1862 A₁₁₉ 1863 L₁₁₉ 1864 V₁₁₉ 1865 A₁₁₈ D₁ 1866 F₁₁₉ 1867 K₁₁₉ 1868 V₉₅ I₂₄ 1869 M₁₁₉ 1870 S₁₁₉ 1871 G₁₁₉ 1872 E₁₀₄ D₁₅ 1873 M₈₂ A₁₇ V₁₂ T₄ L₂ R₁ I₁ 1874 P₁₁₉ 1875 S₁₁₈ A₁ 1876 T₁₀₃ A₁₄ P₁ S₁ 1877 E₁₁₉ 1878 D₁₁₈ E₁ 1879 L₁₀₉ M₈ R₁ I₁ 1880 V₁₁₃ I₅ D₁ 1881 N₁₁₉ 1882 L₁₁₈ M₁ 1883 L₁₁₈ I₁ 1884 P₁₁₇ H₁ L₁ 1885 A₁₁₉ 1886 I₁₁₈ V₁ 1887 L₁₁₈ F₁ 1888 S₁₁₉ 1889 P₁₁₉ 1890 G₁₁₉ 1891 A₁₁₉ 1892 L₁₁₉ 1893 V₁₁₉ 1894 V₁₁₉ 1895 G₁₁₉ 1896 V₁₁₄ I₅ 1897 V₁₁₉ 1898 C₁₁₉ 1899 A₁₁₉ 1900 A₁₁₉ 1901 I₁₁₈ V₁ 1902 L₁₁₈ Q₁ 1903 R₁₁₉ 1904 R₁₁₈ G₁ 1905 H₁₁₉ 1906 V₁₁₉ 1907 G₁₁₈ D₁ 1908 P₁₁₈ X₁ 1909 G₁₁₈ 1910 E₁₁₈ 1911 G₁₁₈ 1912 A₁₁₈ 1913 V₁₁₈ 1914 Q₁₁₇ H₁ 1915 W₁₁₈ 1916 M₁₁₇ V₁ 1917 N₁₁₈ 1918 R₁₁₈ 1919 L₁₁₈ 1920 I₁₁₈ 1921 A₁₁₈ 1922 F₁₁₉ 1923 A₁₁₉ 1924 S₁₁₈ F₁ 1925 R₁₁₈ A₁ 1926 G₁₁₉ 1927 N₁₁₉ 1928 H₁₁₉ 1929 V₁₁₈ D₁ 1930 S₁₁₉ 1931 P₁₁₉ 1932 T₁₁₆ R₂ A₁ 1933 H₁₁₉ 1934 Y₁₁₉ 1935 V₁₁₉ 1936 P₁₁₉ 1937 E₁₁₉ 1938 S₁₁₈ N₁ 1939 D₁₁₇ E₂ 1940 A₁₁₇ P₂ 1941 A₁₁₉ 1942 A₁₀₅ V₁₁ S₁ G₁ Q₁ 1943 R₁₁₈ P₁ 1944 V₁₁₉ 1945 T₁₁₉ 1946 Q₁₁₉ 1947 I₁₁₅ V₄ 1948 L₁₁₉ 1949 S₁₁₉ 1950 S₁₀₄ N₁₁ G₄ 1951 L₁₁₉ 1952 T₁₁₉ 1953 I₁₁₇ V₂ 1954 T₁₁₉ 1955 Q₁₁₉ 1956 L₁₁₉ 1957 L₁₁₉ 1958 K₁₁₁ R₈ 1959 R₁₁₈ K₁ 1960 L₁₁₉ 1961 H₁₁₉ 1962 Q₁₁₇ R₁ H₁ 1963 W₁₁₉ 1964 I₁₁₉ 1965 N₁₁₈ S₁ 1966 E₁₁₉ 1967 D₁₁₉ 1968 C₁₁₉ 1969 S₁₁₉ 1970 T₁₁₉ X₁ 1971 P₁₁₉ M₄ 1972 C₁₁₉ G₄ M₁ 1973 S₂₃₆ A₁ 1974 G₂₃₃ S₃ D₁ 1975 S₂₃₆ T₁ 1976 W₂₃₇ 1977 L₂₃₇ 1978 R₂₂₈ K₉ 1979 D₂₃₄ E₃ 1980 V₂₂₂ I₁₅ 1981 W₂₃₇ 1982 D₂₃₅ E₂ 1983 W₂₃₇ 1984 I₂₃₄ V₃ 1985 D₂₃₇ 1986 T₂₂₉ S₂ A₂ M₂ I₁ X₁ 1987 V₂₃₅ A₁ M₁ 1988 L₂₃₆ V₁ 1989 T₁₇₁ S₃₃ A₂₈ V₂ I₂ N₁ 1990 D₂₃₇ 1991 F₂₃₅ L₂ 1992 K₂₃₆ R₁ 1993 T₂₃₄ N₁ S₁ A₁ 1994 W₂₃₆ R₁ 1995 L₂₃₆ I₁ 1996 Q₂₃₀ K₅ R₂ 1997 S₂₃₁ T₆ 1998 K₂₃₄ R₃ 1999 L₂₃₃ V₃ I₁ 2000 L₂₂₇ M₉ V₁ 2001 P₂₃₆ X₁ 2002 R₂₂₂ Q₁₀ K₄ L₁ 2003 L₂₂₈ M₆ X₁ I₁ F₁ 2004 P₂₃₇ 2005 G₂₃₇ 2006 V₂₁₅ I₁₅ L₅ A₂ 2007 P₂₃₇ 2008 F₂₃₆ X₁ 2009 F₁₁₅ L₁₀₉ I₈ Y₄ M₁ 2010 S₂₃₅ X₂ 2011 C₂₃₆ W₁ 2012 Q₂₃₇ 2013 R₂₃₇ 2014 G₂₃₇ 2015 Y₂₃₆ F₁ 2016 K₁₉₀ R₄₇ 2017 G₂₃₇ 2018 V₂₀₀ I₃₆ A₁ 2019 W₂₃₇ 2020 R₂₃₃ Q₃ L₁ 2021 G₂₃₄ E₂ S₁ 2022 D₂₃₅ E₂ 2023 G₂₃₇ 2024 I₂₁₄ V₂₃ 2025 M₂₃₆ V₁ 2026 Q₁₅₄ H₇₀ Y₈ L₂ N₁ X₁ C₁ 2027 T₂₃₅ A₂ 2028 T₂₂₆ I₈ V₁ A₁ N₁ 2029 C₂₃₇ 2030 P₂₂₅ S₅ Q₃ L₃ A₁ 2031 C₂₃₇ 2032 G₂₃₄ X₂ A₁ 2033 A₂₃₃ G₃ 2034 Q₂₃₁ R₂ H₁ D₁ E₁ 2035 I₂₃₃ L₂ M₁ 2036 T₁₉₂ A₃₂ S₁₂ 2037 G₂₃₆ 2038 H₂₃₆ 2039 V₂₃₅ F₁ 2040 K₂₃₄ R₁ T₁ 2041 N₂₃₄ T₂ 2042 G₂₃₆ 2043 S₂₃₆ 2044 M₂₃₆ 2045 R₂₂₆ K₁₀ 2046 I₂₃₁ X₃ L₁ V₁ 2047 V₂₁₅ A₈ T₆ F₂ I₂ X₁ Y₁ W₁ 2048 G₂₃₆ 2049 P₂₃₃ S₂ X₁ 2050 K₁₄₇ R₈₉ 2051 T₂₂₇ A₈ S₁ 2052 C₂₃₆ 2053 S₂₃₆ 2054 N₂₃₅ S₁ 2055 T₂₁₆ M₁₇ X₁ V₁ A₁ 2056 W₂₃₃ C₃ 2057 H₂₁₅ Y₈ L₃ C₃ R₂ Q₂ S₁ N₁ D₁ 2058 G₂₃₆ 2059 T₂₃₁ A₃ X₁ S₁ 2060 F₂₃₆ 2061 P₂₃₄ S₂ 2062 I₂₃₁ V₄ T₁ 2063 N₂₃₆ 2064 A₂₂₉ T₅ V₁ G₁ 2065 Y₂₂₄ H₁₁ C₁ 2066 T₂₃₆ 2067 T₂₃₆ 2068 G₂₃₅ D₁ 2069 P₂₂₆ S₈ R₁ H₁ 2070 C₂₂₈ S₇ W₁ 2071 T₂₂₇ S₄ V₃ M₁ A₁ 2072 P₂₃₄ X₁ A₁ 2073 S₂₂₀ T₁₃ A₃ 2074 P₂₃₆ 2075 A₂₃₅ T₁ 2076 P₂₃₂ X₃ S₁ 2077 N₂₃₂ S₂ X₁ D₁ 2078 Y₂₃₆ 2079 S₂₂₉ T₅ F₂ 2080 R₂₀₁ K₃₃ N₁ T₁ 2081 A₂₃₄ G₂ 2082 L₂₃₆ 2083 W₂₃₅ L₁ 2084 R₂₃₅ W₁ 2085 V₂₃₄ M₁ R₁ 2086 A₂₂₄ T₇ S₃ V₂ 2087 A₂₃₃ S₁ P₁ F₁ 2088 E₂₃₅ D₁ 2089 E₂₃₄ G₁ D₁ 2090 Y₂₃₆ 2091 V₂₃₃ L₃ 2092 E₂₃₆ 2093 V₂₂₆ I₁₀ 2094 T₂₂₉ R₃ K₂ A₁ V₁ 2095 R₂₃₀ Q₆ 2096 V₂₃₁ M₃ L₂ 2097 G₂₃₆ 2098 D₂₃₂ E₄ 2099 F₂₃₁ S₃ C₁ Y₁ 2100 H₂₃₆ 2101 Y₂₃₅ H₁ 2102 V₂₃₁ I₅ 2103 T₂₃₆ 2104 G₂₃₃ D₂ S₁ 2105 M₂₂₄ V₇ I₄ L₁ 2106 T₂₃₆ 2107 T₂₂₄ A₇ N₂ V₁ I₁ S₁ 2108 D₂₃₆ 2109 N₂₃₅ D₁ 2110 V₁₈₂ I₄₄ L₁₀ 2111 K₂₃₀ R₃ X₂ E₁ 2112 C₂₃₆ 2113 P₂₃₆ 2114 C₂₃₅ X₁ 2115 Q₂₃₆ 2116 V₂₃₅ E₁ 2117 P₂₃₆ 2118 A₂₃₀ T₂ P₂ S₂ 2119 P₂₃₄ X₁ L₁ 2120 E₂₃₆ D₁ 2121 F₂₃₆ X₁ 2122 F₂₃₆ 2123 T₂₃₄ K₁ S₁ 2124 E₂₃₄ X₁ A₁ 2125 V₁₈₇ L₄₉ 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2250 V₂₈₈ I₉ T₁ 2251 V₂₈₄ I₁₃ X₁ 2252 I₂₈₁ V₁₇ 2253 L₂₉₅ M₂ X₁ 2254 D₂₉₈ 2255 S₂₉₇ X₁ 2256 F₂₉₅ X₂ L₁ 2257 D₂₁₈ E₇₄ V₄ G₁ C₁ 2258 P₂₉₆ Q₁ S₁ 2259 L₂₉₁ I₄ V₁ P₁ C₁ 2260 R₂₇₅ Q₁₃ H₆ V₂ G₁ Y₁ 2261 A₂₉₂ V₄ T₂ 2262 E₂₉₄ A₁ D₁ G₁ V₁ 2263 E₂₉₀ K₂ X₂ G₂ D₂ R₁ 2264 D₂₈₄ E₈ G₅ S₁ N₁ 2265 E₂₇₉ D₉ V₅ G₄ Q₂ 2266 R₂₇₁ G₁₉ K₆ W₂ N₁ 2267 E₂₉₉ 2268 V₂₁₈ I₆₂ M₈ P₈ E₁ R₁ L₁ 2269 S₂₉₉ 2270 V₂₈₂ I₁₃ L₂ X₁ A₁ 2271 P₂₁₈ A₆₄ E₁₆ T₁ 2272 A₂₉₆ S₂ E₁ 2273 E₂₉₆ X₂ D₁ 2274 I₂₉₅ V₃ X₁ 2275 L₂₉₈ P₁ 2276 R₂₈₈ L₇ Q₂ W₂ 2277 K₂₃₀ R₆₈ T₁ 2278 S₂₃₀ T₆₀ P₈ N₁ 2279 R₂₄₄ K₅₃ G₂ 2280 K₂₆₂ R₂₅ N₃ E₃ X₁ D₁ A₁ V₁ S₁ G₁ 2281 F₂₉₈ Y₁ 2282 P₂₉₆ X₂ T₁ 2283 P₁₆₉ R₇₀ S₂₇ A₁₆ L₈ Q₆ T₂ M₁ 2284 A₂₉₈ X₁ 2285 M₁₈₆ L₉₂ I₁₈ V₃ 2286 P₂₉₈ L₁ 2287 I₂₂₈ V₇₀ E₁ 2288 W₂₉₉ 2289 A₂₉₇ T₁ X₁ 2290 R₂₉₅ P₂ H₁ Q₁ 2291 P₂₉₈ A₁ 2292 D₂₉₄ E₃ G₁ N₁ 2293 Y₂₉₈ H₁ 2294 N₂₉₈ Y₁ 2295 P₂₉₉ 2296 P₂₉₉ 2297 L_(297.) I₁ V₁ 2298 L₂₂₄ I₄₀ V₃₄ M₁ 2299 E₂₉₅ Q₄ 2300 S₂₆₃ P₂₈ T₄ A₃ X₁ 2301 W₂₉₉ 2302 K₂₈₆ R₁₂ E₁ 2303 D₂₅₅ N₂₄ A₆ K₅ S₅ R₂ G₂ 2304 P₂₉₉ 2305 D₂₈₅ A₅ E₄ N₄ G₁ 2306 Y₂₉₈ S₁ 2307 V₂₇₆ I₈ A₅ T₄ D₂ E₁ S₁ N₁ P₁ 2308 P₂₉₈ L₁ 2309 P₂₉₇ X₂ 2310 V₂₉₂ A₅ G₁ L₁ 2311 V₂₉₈ X₁ 2312 H₂₉₆ R₁ Y₁ L₁ 2313 G₂₉₈ 2314 C₂₉₈ 2315 P₂₉₇ A₁ 2316 L₂₉₇ F₁ 2317 P₂₉₇ S₁ 2318 P₂₉₁ S₇ 2319 T₂₀₉ A₆₃ V₁₃ I₈ P₂ N₁ S₁ D₁ 2320 K₂₆₅ R₁₈ T₈ E₃ G₃ 2321 A₂₅₅ T₂₀ G₁₀ V₇ N₁ I₁ E₁ S₁ D₁ 2322 P₂₉₂ A₃ H₁ L₁ 2323 P₂₉₇ 2324 I₂₆₆ V₂₈ L₂ T₁ 2325 P₂₉₅ L₁ S₁ 2326 P₂₉₇ 2327 P₂₉₆ Q₁ 2328 R₂₉₆ X₁ 2329 R₂₇₄ K₂₃ 2330 K₂₉₄ R₃ 2331 R₂₈₃ K₁₄ 2332 T₂₉₃ A₂ V₁ 2333 V₂₈₃ I₉ X₁ F₁ 2334 V₂₈₂ I₁₀ A₁ 2335 L₂₉₃ 2336 T₂₈₂ S₁₁ 2337 E₂₈₈ D₄ G₁ 2338 S₂₉₃ 2339 T₂₇₈ N₈ S₆ A₁ 2340 V₂₈₈ L₃ M₂ 2341 S₂₉₁ P₁ A₁ 2342 S₂₉₁ T₁ A₁ 2343 A₂₈₈ V₄ S₁ 2344 L₂₉₃ 2345 A₂₉₃ 2346 E₂₉₁ G₁ D₁ 2347 L₂₉₁ F₂ 2348 A₂₉₀ X₁ P₁ G₁ 2349 T₂₇₂ A₁₀ V₅ K₃ I₃ 2350 K₂₈₇ R₂ E₁ T₁ S₁ Q₁ 2351 T₂₈₇ A₅ S₁ 2352 F₂₉₃ 2353 G₂₅₉ S₃₁ D₂ C₁ 2354 S₂₆₄ G₂₀ N₇ D₂ 2355 S₂₉₀ P₂ F₁ 2356 E₁₅₂ G₁₂₈ K₅ D₃ T₁ R₁ X₁ M₁ V₁ 2357 S₂₈₂ P₄ T₃ L₃ A₁ 2358 S₂₇₈ P₆ A₃ T₂ L₂ R₁ E₁ 2359 A₂₇₀ G₉ I₅ S₃ T₃ V₂ C₁ 2360 V₂₂₂ A₅₂ I₁₀ G₄ X₁ D₁ S₁ P₁ T₁ 2361 D₂₈₇ G₃ A₂ N₁ 2362 S₂₈₅ G₄ N₃ R₁ 2363 G₂₈₇ S₅ R₁ 2364 T₂₇₅ A₆ V₆ M₅ I₁ 2365 A₂₈₃ V₅ S₂ E₁ T₁ M₁ 2366 T₂₆₄ S₂₀ A₄ I₄ P₁ 2367 A₂₇₄ G₁₄ T₃ D₁ I₁ 2368 P₂₇₃ S₁₃ L₅ Y₁ C₁ 2369 P₂₈₄ H₄ L₄ S₁ 2370 D₂₆₅ G₂₀ N₃ E₃ T₂ 2371 Q₂₈₂ L₄ R₂ E₂ H₂ G₁ 2372 P₁₆₂ A₇₃ S₂₃ T₁₉ L₁₂ V₃ I₁ 2373 S₂₅₇ P₁₉ L₈ F₆ A₁ Y₁ T₁ 2374 D₂₄₂ N₁₉ G₁₄ E₆ S₅ A₅ K₂ 2375 D₂₁₀ N₄₈ G₁₀ E₉ S₆ A₅ C₃ V₁ Y₁ 2376 G₂₇₈ D₁₂ E₁ S₁ V₁ 2377 D₂₄₅ G₄₃ N₂ A₁ H₁ E₁ 2378 A₁₂₄ T₁₀₉ K₄₄ R₅ S₄ P₁ M₁ G₁ V₁ L₁ N₁ Q₁ 2379 G₂₅₅ E₂₈ R₄ S₂ C₁ D₁ A₁ K₁ 2380 S₂₉₀ T₃ 2381 D₂₉₃ 2382 V₂₂₉ A₅₈ I₄ G₁ T₁ 2383 E₂₄₅ G₄₈ 2384 S₂₉₁ A₁ E₁ 2385 Y₂₇₆ C₈ H₆ F₂ A₁ 2386 S₂₉₂ P₁ 2387 S₂₉₃ 2388 M₂₉₁ T₂ 2389 P₂₉₃ 2390 P₂₉₃ 2391 L₂₉₃ 2392 E₂₉₃ 2393 G₂₉₃ 2394 E₂₉₃ 2395 P₂₉₂ Q₁ 2396 G₂₉₃ 2397 D₂₉₂ T₁ 2398 P₂₉₂ L₁ 2399 D₂₉₃ 2400 L₂₈₉ F₄ 2401 S₂₉₁ N₂ 2402 D₂₉₂ E₁ 2403 G₂₃₆ 2404 S₂₃₆ 2405 W₂₃₄ G₂ 2406 S₂₃₆ X₁ 2407 T₂₃₆ S₁ 2408 V₂₂₉ M₈ 2409 S₂₃₅ G₁ N₁ 2410 E₂₂₆ G₁₀ D₁ 2411 E₂₃₄ X₁₀ G₁ Q₁ 2412 A₂₃₂ D₅ V₄ G₃ P₂ 2413 S₁₆₈ G₇₀ T₂ N₂ E₁ D₁ A₁ R₁ 2414 E₂₃₆ D₅ Q₄ G₁ 2415 D₂₃₈ S₂ V₂ N₂ G₂ 2416 V₂₄₁ I₅ A₁ X₁ 2417 V₂₄₅ I₃ 2418 C₂₄₈ 2419 C₂₄₄ W₃ X₁ 2420 S₁₇₀ P₂ X₁ 2421 M₁₇₁ L₁ 2422 S₁₇₂ 2423 Y₁₆₆ H₁ S₁ 2424 T₁₅₅ S₁₀ A₁ P₁ 2425 W₁₆₇ 2426 T₁₆₆ A₁ 2427 G₁₆₇ 2428 A₁₆₇ 2429 L₁₆₆ M₁ 2430 I₁₆₄ V₂ N₁ 2431 T₁₆₆ P₁ 2432 P₁₆₇ 2433 C₁₆₇ 2434 A₁₃₅ S₃₁ G₁ 2435 A₁₆₇ 2436 E₁₆₇ 2437 E₁₆₅ G₂ 2438 S₁₅₉ T₅ N₃ 2439 K₁₆₅ Q₁ E₁ 2440 L₁₆₇ 2441 P₁₆₇ 2442 I₁₆₇ 2443 N₁₆₆ I₁ 2444 A₁₂₈ P₃₈ S₁ 2445 L₁₆₇ 2446 S₁₆₅ I₁ N₁ 2447 N₁₆₆ Y₁ 2448 S₁₆₂ P₄ T₁ 2449 L₁₆₆ Q₁ 2450 L₁₆₇ 2451 R₁₆₇ 2452 H₁₆₃ N₃ R₁ 2453 H₁₆₆ R₁ 2454 N₁₆₄ S₃ 2455 M₁₅₉ L₈ 2456 V₁₆₂ I₅ 2457 Y₁₆₇ 2458 A₁₅₇ S₈ T₁ V₁ 2459 T₁₆₆ A₁ 2460 T₁₆₇ 2461 S₁₆₇ 2462 R₁₆₇ 2463 S₁₆₆ I₁ 2464 A₁₆₇ 2465 S₁₃₆ G₂₆ C₄ V₁ 2466 Q₁₀₇ L₆₀ 2467 R₁₆₇ 2468 Q₁₆₇ 2469 K₁₆₂ R₅ 2470 K₁₆₇ 2471 V₁₆₇ 2472 T₁₆₆ A₁ 2473 F₁₆₅ I₂ 2474 D₁₆₇ 2475 R₁₆₇ 2476 L₁₅₃ M₉ Q₅ 2477 Q₁₆₇ 2478 V₁₆₇ 2479 L₁₆₆ Q₁ 2480 D₁₆₆ E₁ 2481 D₁₆₂ N₃ S₁ K₁ 2482 H₁₆₇ 2483 Y₁₆₇ 2484 R₁₅₉ Q₇ W₁ 2485 D₁₆₆ G₁ 2486 V₁₆₇ 2487 L₁₆₇ 2488 K₁₆₇ 2489 E₁₆₄ D₂ G₁ 2490 M₁₆₃ V₃ I₁ 2491 K₁₆₆ N₁ 2492 A₁₆₄ V₃ 2493 K₁₆₆ E₁ 2494 A₁₆₇ 2495 S₁₆₇ 2496 T₁₆₆ K₁ 2497 V₁₆₇ 2498 K₁₆₃ R₂ N₁ 2499 A₁₆₆ 2500 K₁₄₄ R₂₂ 2501 L₁₆₅ F₁ 2502 L₁₆₆ 2503 S₁₆₃ P₂ T₁ 2504 V₁₂₄ I₄₁ L₁ 2505 E₁₆₆ 2506 E₁₆₅ X₁ 2507 A₁₆₅ P₁ 2508 C₁₆₆ 2509 K₁₃₆ M₂₂ R₆ Q₁ N₁ 2510 L₁₆₅ W₁ 2511 T₁₆₆ 2512 P₁₆₆ 2513 P₁₆₆ 2514 H₁₆₄ Q₁ L₁ 2515 S₁₆₆ 2516 A₁₆₆ 2517 K₁₀₈ R₅₇ S₁ 2518 S₁₆₅ L₁ 2519 K₁₆₃ Q₃ 2520 F₁₅₆ Y₈ V₁ S₁ 2521 G₁₆₆ 2522 Y₁₆₆ X₁ 2523 G₁₆₇ 2524 A₁₆₆ G₁ 2525 K₁₆₆ S₁ 2526 D₁₆₇ 2527 V₁₆₇ 2528 R₁₆₇ 2529 N₁₃₆ S₃₁ 2530 L₁₆₇ 2531 S₁₆₄ T₃ 2532 S₁₄₈ G₁₃ N₃ R₃ 2533 K₁₃₂ R₃₄ G₁ 2534 A₁₆₇ 2535 V₁₂₁ I₃₇ T₈ L₁ 2536 N₁₅₂ D₅ K₃ S₃ R₂ T₂ 2537 H₁₆₇ 2538 I₁₆₇ 2539 R₁₁₇ H₃₉ N₆ S₂ L₂ T₁ 2540 S₁₆₇ 2541 V₁₆₇ 2542 W₁₆₇ 2543 K₁₃₄ E₃₃ 2544 D₁₆₇ 2545 L₁₆₆ S₁ 2546 L₁₆₅ Q₂ 2547 E₁₆₆ D₁ 2548 D₁₆₇ 2549 T₁₄₇ S₉ N₈ P₂ D₁ 2550 E₁₃₃ V₁₂ D₁₂ Q₆ A₂ K₁ I₁ 2551 T₁₆₇ 2552 P₁₆₇ 2553 I₁₆₁ L₅ F₁ 2554 D₁₃₈ N₁₇ Q₄ S₃ E₂ T₁ I₁ P₁ 2555 T₁₆₇ 2556 T₁₆₆ V₁ 2557 I₁₅₁ V₁₆ 2558 M₁₆₇ 2559 A₁₆₇ 2560 K₁₆₇ 2561 N₁₄₄ S₂₃ 2562 E₁₆₇ 2563 V₁₆₂ I₅ 2564 F₁₆₆ I₁ 2565 C₁₆₄ V₂ D₁ 2566 V₁₆₁ I₅ L₁ 2567 Q₁₆₄ D₁ K₁ E₁ 2568 P₁₆₇ 2569 E₁₆₅ T₁ X₁ 2570 K₁₆₅ M₁ R₁ 2571 G₁₆₇ 2572 G₁₆₇ 2573 R₁₆₇ 2574 K₁₆₇ 2575 P₁₆₀ A₅ S₂ 2576 A₁₆₇ 2577 R₁₆₇ 2578 L₁₆₃ F₄ 2579 I₁₆₇ 2580 V₁₆₇ 2581 F₁₆₄ Y₃ 2582 P₁₆₇ 2583 D₁₆₇ 2584 L₁₆₇ 2585 G₁₆₇ 2586 V₁₆₇ 2587 R₁₆₇ 2588 V₁₆₆ I₁ 2589 C₁₆₇ 2590 E₁₆₇ 2591 K₁₆₇ 2592 M₁₆₇ 2593 A₁₆₇ 2594 L₁₆₇ 2595 Y₁₆₇ 2596 D₁₆₆ N₁ 2597 V₁₆₆ X₁ 2598 V₁₆₇ 2599 S₁₆₇ 2600 T₁₆₃ N₃ I₁ 2601 L₁₆₇ 2602 P₁₆₇ 2603 Q₁₅₉ H₆ K₁ R₁ 2604 A₁₆₃ V₂ P₁ T₁ 2605 V₁₆₇ 2606 M₁₆₇ 2607 G₁₆₇ 2608 S₁₅₀ P₁₃ A₄ 2609 S₁₆₁ A₅ L₁ 2610 Y₁₆₆ F₁ 2611 G₁₆₇ 2612 F₁₆₆ C₁ 2613 Q₁₆₇ 2614 Y₁₆₇ 2615 S₁₆₇ 2616 P₁₆₅ L₁ S₁ 2617 G₁₄₃ K₂₁ A₂ S₁ 2618 Q₁₆₆ H₁ 2619 R₁₆₇ 2620 V₁₆₇ 2621 E₁₆₃ D₃ Q₁ 2622 F₁₆₇ 2623 L₁₆₇ 2624 V₁₆₆ L₁ 2625 N₁₅₅ K₁₁ D₁ 2626 A₁₂₉ T₃₈ 2627 W₁₆₇ 2628 K₁₆₆ S₁ 2629 S₁₃₁ K₂₉ A₅ Q₂ 2630 K₁₆₇ 2631 K₁₅₈ R₇ N₁ E₁ 2632 N₆₆ C₅₇ S₃₄ T₆ V₂ A₁ K₁ 2633 P₁₆₆ A₁ 2634 M₁₆₇ 2635 G₁₆₅ A₁ X₁ 2636 F₁₆₅ X₁ S₁ 2637 A₉₆ S₇₁ 2638 Y₁₆₇ 2639 D₁₆₇ 2640 T₁₆₆ A₁ 2641 R₁₆₇ 2642 C₁₆₆ R₁ 2643 F₁₆₇ 2644 D₁₆₆ E₁ 2645 S₁₆₇ X₁ 2646 T₁₆₈ 2647 V₁₆₈ 2648 T₁₆₈ X₂ 2649 E₁₆₉ Q₁ 2650 N₈₉ S₈₀ H₁ 2651 D₁₇₀ 2652 I₁₇₀ 2653 R₁₇₀ 2654 V₁₄₀ T₂₄ I₅ F₁ 2655 E₁₆₉ G₁ 2656 E₁₇₀ 2657 S₁₆₉ P₁ 2658 I₁₇₀ 2659 Y₁₇₀ 2660 Q₁₇₀ 2661 C₁₅₆ S₁₄ 2662 C₁₇₀ X₁₃ 2663 D₁₈₂ E₁ 2664 L₁₈₃ 2665 A₁₇₄ V₃ D₃ T₁ S₁ G₁ 2666 P₁₈₃ 2667 E₁₈₀ D₃ X₁ 2668 A₁₈₄ 2669 R₁₆₄ K₁₉ S₁ 2670 Q₁₆₈ L₁₀ R₃ K₂ T₁ 2671 A₁₆₅ V₁₉ 2672 I₁₈₄ 2673 R₁₃₉ K₄₅ 2674 S₁₈₄ 2675 L₁₈₄ 2676 T₁₈₄ 2677 E₁₈₄ 2678 R₁₈₄ 2679 L₁₈₄ 2680 Y₁₈₄ 2681 I₁₆₂ V₂₂ 2682 G₁₈₄ 2683 G₁₈₄ 2684 P₁₈₄ 2685 L₁₇₇ M₇ 2686 T₁₈₃ I₁ 2687 N₁₈₄ 2688 S₁₈₄ 2689 K₁₈₄ 2690 G₁₈₄ 2691 Q₁₈₄ 2692 N₁₇₃ S₈ D₂ H₁ 2693 C₁₈₄ 2694 G₁₈₄ 2695 Y₁₈₄ 2696 R₁₈₄ 2697 R₁₈₄ 2698 C₁₈₄ 2699 R₁₈₄ 2700 A₁₈₁ V₂ X₁ 2701 S₁₈₃ T₁ 2702 G₁₈₃ V₁ 2703 V₁₈₄ 2704 L₁₈₄ 2705 T₁₈₄ R₁ 2706 T₁₈₄ R₁ 2707 S₁₈₄ R₁ 2708 C₁₈₄ X₁ 2709 G₁₈₃ S₁ A₁ 2710 N₁₈₄ X₁ 2711 T₁₈₄ X₁ 2712 L₁₈₂ I₂ X₁ 2713 T₁₈₅ 2714 C₁₈₅ 2715 Y₁₈₂ F₂ H₁ 2716 L₁₈₅ 2717 K₁₈₅ 2718 A₁₈₅ 2719 S₁₄₉ T₃₀ A₆ 2720 A₁₈₅ 2721 A₁₈₅ 2722 C₁₈₅ 2723 R₁₈₅ 2724 A₁₈₃ D₁ V₁ 2725 A₁₈₄ G₁ 2726 K₁₈₃ G₁ X₁ 2727 L₁₈₄ X₁ 2728 Q₁₆₅ R₂₀ 2729 D₁₈₃ G₁ S₁ 2730 C₁₈₄ Y₁ 2731 T₁₈₅ 2732 M₁₈₂ L₂ T₁ 2733 L₁₈₅ 2734 V₁₈₅ 2735 C₁₁₉ N₆₅ H₁ 2736 G₁₈₅ 2737 D₁₈₅ 2738 D₁₈₅ 2739 L₁₈₄ X₁ 2740 V₁₈₂ I₂ X₁ 2741 V₁₈₃ X₁ I₁ 2742 I₁₈₄ X₁ 2743 C₁₈₄ X₁ 2744 E₁₇₉ D₅ X₁ 2745 S₁₈₃ X₁ C₁ 2746 A₁₈₁ E₂ T₁ X₁ 2747 G₁₈₄ 2748 T₁₈₁ A₁ V₁ I₁ 2749 Q₁₈₁ E₂ X₁ 2750 E₁₈₄ 2751 D₁₈₄ 2752 A₁₆₉ E₁₄ P₁ 2753 A₁₈₃ E₁ 2754 S₁₅₁ N₂₆ A₄ R₂ C₁ 2755 L₁₈₄ 2756 R₁₈₄ 2757 V₁₆₃ A₂₀ F₁ 2758 F₁₈₄ 2759 T₁₈₃ A₁ 2760 E₁₈₄ 2761 A₁₈₄ 2762 M₁₈₄ 2763 T₁₈₄ 2764 R₁₈₄ 2765 Y₁₇₆ N₇ X₁ 2766 S₁₈₂ X₁ 2767 A₁₈₂ G₁ 2768 P₁₇₆ H₇ 2769 P₁₇₅ G₅ A₂ X₁ 2770 G₁₇₄ X₄ H₃ A₁ S₁ 2771 D₁₇₄ X₅ G₁ K₁ E₁ S₁ 2772 P₁₆₂ L₁₈ C₂ S₁ 2773 P₁₇₉ V₁ L₁ 2774 Q₁₁₇ K₄₇ R₁₆ X₂ 2775 P₁₈₁ 2776 E₁₇₆ A₃ V₁ T₁ 2777 Y₁₈₁ 2778 D₁₈₁ 2779 L₁₇₉ Q₁ K₁ 2780 E₁₈₁ 2781 L₁₇₆ S₂ X₁ R₁ M₁ 2782 I₁₈₁ 2783 T₁₇₉ I₁ S₁ 2784 S₁₈₀ A₁ 2785 C₁₈₀ R₁ 2786 S₁₇₃ X₈ 2787 S₁₇₂ P₁ 2788 N₁₇₃ 2789 V₁₇₃ 2790 S₁₇₃ 2791 V₁₇₃ 2792 A₁₇₁ X₂ 2793 H₁₇₂ Y₁ 2794 D₁₇₃ 2795 A₁₇₁ V₂ 2796 S₁₆₆ A₃ L₂ T₁ N₁ 2797 G₁₇₁ N₂ 2798 K₁₇₁ R₂ 2799 R₁₇₂ X₁ 2800 V₁₇₃ 2801 Y₁₇₃ 2802 Y₁₇₃ 2803 L₁₇₃ 2804 T₁₇₃ 2805 R₁₇₃ 2806 D₁₇₁ N₂ 2807 P₁₇₃ 2808 T₁₆₁ A₅ I₄ S₂ X₁ 2809 T₁₆₆ I₅ N₁ S₁ 2810 P₁₇₂ X₁ 2811 L₁₅₅ I₁₄ F₄ 2812 A₁₆₉ S₂ G₂ 2813 R₁₇₃ 2814 A₁₇₁ X₁ T₁ 2815 A₁₇₃ 2816 W₁₇₃ 2817 E₁₇₂ A₁ 2818 T₁₇₂ A₁ 2819 A₁₆₇ V₄ S₁ X₁ 2820 R₁₆₇ K₆ 2821 H₁₆₈ S₅ 2822 T₁₇₂ I₁ 2823 P₁₇₁ L₁ S₁ 2824 V₁₆₇ I₄ T₂ 2825 N₁₇₃ 2826 S₁₇₂ T₁ 2827 W₁₇₃ 2828 L₁₇₃ 2829 G₁₇₃ 2830 N₁₇₃ 2831 I₁₇₂ V₁ 2832 I₁₇₂ M₁ 2833 M₁₇₃ 2834 Y₁₇₂ F₁ 2835 A₁₇₂ G₁ 2836 P₁₇₃ 2837 T₁₇₂ A₁ 2838 L₁₇₂ I₁ 2839 W₁₇₃ 2840 A₁₆₂ V₁₁ 2841 R₁₇₃ 2842 M₁₇₃ 2843 I₁₅₆ V₁₇ 2844 L₁₆₈ M₃ I₂ 2845 M₁₇₁ L₂ 2846 T₁₇₀ P₂ I₁ 2847 H₁₇₃ 2848 F₁₇₁ I₂ 2849 F₁₇₂ S₁ 2850 S₁₇₃ 2851 I₁₇₁ V₁ N₁ 2852 L₁₇₂ X₁ 2853 L₁₇₁ M₁ I₁ 2854 A₁₆₇ V₃ X₁ P₁ F₁ 2855 Q₁₇₂ R₁ 2856 E₁₇₃ 2857 Q₁₇₂ Y₁ 2858 L₁₇₃ 2859 E₁₆₁ G₈ D₄ 2860 K₁₇₀ R₂ Q₁ 2861 A₁₆₅ T₆ V₂ 2862 L₁₇₀ X₂ Q₁ 2863 D₁₆₆ E₅ X₂ 2864 C₁₇₂ F₁ 2865 Q₁₇₂ H₁ 2866 I₁₇₃ 2867 Y₁₇₃ 2868 G₁₇₁ R₁ E₁ 2869 A₁₇₂ T₁ 2870 C₁₁₆ T₃₈ Y₈ I₅ X₂ S₂ H₁ V₁ 2871 Y₁₇₀ H₂ 2872 S₁₇₂ 2873 I₁₇₀ V₁ 2874 E₁₅₅ Q₃ G₁ 2875 P₁₅₉ 2876 L₁₅₉ 2877 D₁₅₉ 2878 L₁₅₈ I₁ 2879 P₁₅₉ 2880 Q₁₅₆ P₂ L₁ 2881 I₁₅₈ V₁ 2882 I₁₅₉ 2883 Q₉₉ E₅₉ G₁ 2884 R₁₅₉ 2885 L₁₅₉ 2886 H₁₅₉ 2887 G₁₅₈ S₁ 2888 L₁₅₉ 2889 S₁₅₈ G₁ 2890 A₁₅₉ 2891 F₁₅₉ 2892 S₁₅₉ 2893 L₁₅₉ 2894 H₁₅₉ 2895 S₁₅₆ N₁ I₁ T₁ 2896 Y₁₅₉ 2897 S₁₅₈ P₁ 2898 P₁₅₉ 2899 G₁₅₈ R₁ 2900 E₁₅₉ 2901 I₁₅₉ 2902 N₁₅₉ 2903 R₁₅₉ 2904 V₁₅₉ 2905 A₁₅₉ 2906 S₁₂₁ A₃₅ T₂ X₁ 2907 C₁₅₈ S₁ 2908 L₁₅₉ 2909 R₁₅₉ 2910 K₁₅₈ Q₁ 2911 L₁₅₉ 2912 G₁₅₉ 2913 V₁₅₉ 2914 P₁₅₉ 2915 P₁₅₉ 2916 L₁₅₉ 2917 R₁₅₉ 2918 V₁₄₆ A₁₀ T₂ I₁ 2919 W₁₅₉ 2920 R₁₅₈ I₁ 2921 H₁₅₈ L₁ 2922 R₁₅₉ 2923 A₁₅₉ 2924 R₁₅₈ K₁ 2925 S₁₅₆ N₁ G₁ R₁ 2926 V₁₅₉ 2927 R₁₅₉ 2928 A₁₅₉ 2929 K₁₄₈ R₁₁ 2930 L₁₅₉ 2931 L₁₅₉ 2932 S₁₅₉ 2933 Q₁₅₂ R₆ P₁ 2934 G₁₅₈ R₁ 2935 G₁₅₉ 2936 R₁₅₇ K₁ E₁ 2937 A₁₅₇ Y₁ G₁ 2938 A₁₅₈ S₁ 2939 T₁₂₄ N₂₆ I₉ 2940 C₁₅₉ 2941 G₁₅₈ X₁ 2942 K₁₃₈ R₁₉ X₁ T₁ 2943 Y₁₅₉ 2944 L₁₅₉ 2945 F₁₅₉ 2946 N₁₅₉ 2947 W₁₅₉ 2948 A₁₅₉ 2949 V₁₅₉ 2950 R₁₁₇ K₄₂ 2951 T₁₅₉ 2952 K₁₅₉ 2953 L₁₅₉ 2954 K₁₅₉ 2955 L₁₅₉ 2956 T₁₅₉ 2957 P₁₅₉ 2958 I₁₅₆ N₃ 2959 P₁₅₉ 2960 A₁₅₃ E₄ G₂ 2961 A₁₅₉ 2962 S₁₅₇ Y₁ F₁ 2963 Q₁₁₀ R₄₅ H₂ K₂ 2964 L₁₅₉ 2965 D₁₅₉ 2966 L₁₅₉ 2967 S₁₅₉ 2968 G₉₃ S₅₁ N₁₃ D₁ K₁ 2969 W₁₅₉ 2970 F₁₅₉ 2971 V₁₄₉ I₆ T₄ 2972 A₁₅₉ 2973 G₁₅₉ 2974 Y₁₅₉ 2975 S₁₄₃ G₁₀ N₆ 2976 G₁₅₉ 2977 G₁₅₉ 2978 D₁₅₉ 2979 I₁₅₅ V₃ T₁ 2980 Y₁₅₉ 2981 H₁₅₈ R₁ 2982 S₁₅₉ 2983 L₁₃₃ V₂₄ P₂ 2984 S₁₅₇ P₂ 2985 R₁₄₇ H₁₀ P₁ C₁ 2986 A₁₅₅ X₂ T₂ 2987 R₁₅₇ 2988 P₁₅₇ 2989 R₁₅₇ 2990 W₁₅₇ 2991 F₁₄₉ L₇ P₁ 2992 M₁₅₃ L₄ 2993 L₈₄ W₇₁ F₂ 2994 C₁₅₇ 2995 L₁₅₇ 2996 L₁₅₅ F₁ P₁ 2997 L₁₅₇ 2998 L₁₅₇ 2999 S₁₅₄ T₁ F₁ X₁ 3000 V₁₅₇ 3001 G₁₅₇ 3002 V₁₅₇ 3003 G₁₅₇ 3004 I₁₅₂ V₅ 3005 Y₁₅₆ N₁ 3006 L₁₅₆ C₁ 3007 L₁₅₆ X₁ 3008 P₁₅₆ 3009 N₁₅₄ A₁ K₁ 3010 R₁₅₆ 

1. A composition comprising at least one nucleic acid sequence which codes for an HCV 1a E2 protein, said protein comprising the consensus sequence at residues 384-746 of SEQ ID NO:
 1. 2. A composition comprising at least one nucleic acid sequence which codes for an immunogenic fragment of an HCV 1a E2 protein, said fragment comprising at least 45 contiguous residues of the consensus sequence at residues 384-746 of SEQ ID NO: 1, and further wherein the immunogenic fragment comprises residues 518-531 of SEQ ID NO:
 1. 